Treatment of hepatitis c virus related diseases using hydroxychloroquine or a combination of hydroxychloroquine and an anti-viral agent

ABSTRACT

Methods of treating a hepatitis C virus (HCV) related disease, such as HCV infections in subjects non-responsive to anti-HCV therapy, are described herein, comprising administering to the subject a therapeutically effective amount of hydroxychloroquine. An antiviral agent may be co-administered with the hydroxychloroquine. Methods utilizing synergistic combinations of hydroxychloroquine and an antiviral agent are disclosed. Further disclosed are compositions comprising hydroxychloroquine and an antiviral agent, as well as hydroxychloroquine and uses thereof for the treatment of a hepatitis C virus (HCV) related disease.

RELATED APPLICATIONS

This application is a Continuation-In-Part (CIP) of PCT PatentApplication No. PCT/IB2011/052762 having International filing date ofJun. 23, 2011, which claims the benefit of priority under 35 USC §119(e)of U.S. Provisional Patent Application No. 61/358,014 filed on Jun. 24,2010. The contents of the above applications are all incorporated byreference as if fully set forth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to therapyand, more particularly, but not exclusively, to methods and compositionsfor the treatment of Hepatitis C Virus (HCV) related diseases such aschronic HCV infection.

HCV is a positive-stranded RNA virus which has been classified as aseparate genus in the Flaviviridae family. All members of theFlaviviridae family have enveloped virions that contain a positivestranded RNA genome encoding all known virus-specific proteins viatranslation of a single, uninterrupted, open reading frame. The singlestrand HCV RNA genome is approximately 9500 nucleotides in length andhas a single open reading frame (ORF) encoding a single largepolyprotein of about 3,000 amino acids. In infected cells, thispolyprotein is cleaved at multiple sites by cellular and viral proteasesto produce the structural and non-structural (NS) proteins. In the caseof HCV, the generation of mature non-structural proteins (NS2, NS3,NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. Thefirst one cleaves at the NS2-NS3 junction; the second one is a serineprotease contained within the N-terminal region of NS3 and mediates allthe subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4Acleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A,NS5A-NS5B sites. The NS4A protein appears to serve multiple functions,acting as a co-factor for the NS3 protease and possibly assisting in themembrane localization of NS3 and other viral replicase components. TheNS3 protein also exhibits nucleoside triphosphatase and RNA helicaseactivities. NS5B is a RNA-dependent RNA polymerase that is involved inthe replication of HCV.

Infection by hepatitis C virus (HCV) is a compelling human medicalproblem. HCV is recognized as the causative agent for most cases ofnon-A, non-B hepatitis, with an estimated human sero-prevalence of 3%globally. Nearly four million individuals may be infected in the UnitedStates alone. Upon first exposure to HCV only about 20% of infectedindividuals develop acute clinical hepatitis while others appear toresolve to the infection spontaneously. In almost 70% of instances,however, the virus establishes a chronic infection that persists fordecades. This usually results in recurrent and progressively worseningliver inflammation, which often leads to more severe disease states suchas cirrhosis and hepatocellular carcinoma.

The combination of a pegylated interferon (e.g., peg-IFN alpha-2a/b) andtwice-daily oral doses of ribavirin, an anti-viral agent, is the currentstandard of care for the treatment of chronic HCV infection. Patientswho will ultimately achieve a sustained virologic response to peg-IFNand ribavirin therapy usually develop a rapid decline in HCV-RNA levelsafter initiation of therapy, with levels becoming undetectable within4-24 weeks. Liver enzyme levels become normal, and histologic findingsimprove markedly. With the above-mentioned combination therapy,approximately 75% to 80% of patients with HCV genotype 2 or 3 infectionand 40% to 50% of those with genotype 1 infection achieve a sustainedvirologic response (SVR) [Sherman K. E., Clinical Need and TherapeuticTargets for New HCV Agents, in The Future of HCV: Small molecules inDevelopment for Chronic Hepatitis C, Clinical Care Options LLC, 2007].

However, success rate of this combined therapy is limited as its outcomeis highly dependent on the infecting HCV genotype. This treatment iseffective in fewer than 50% of patients infected with HCV genotype 1 or4, the most represented genotypes in Europe and USA. In many cases,non-response is related to host or viral factors that impair activationof the host's innate, interferon-driven immune response.

Others may achieve viral reduction during therapy but cannot toleratefull therapeutic doses or an adequate duration of treatment because ofcytopenia, fatigue, or other adverse effects of treatment. Indeed, dosemodifications for these reasons are required in 35% to 42% of treatedpatients, and approximately one third of these patients eventuallydiscontinue treatment altogether. These dose reductions, temporaryinterruptions, and aborted treatment courses reduce the chance ofachieving SVR.

Finally, the combination of peg-IFN and ribavirin is contraindicatedaltogether in many patients who are in need of anti-HCV therapy.Contraindications for therapy include severe cytopenia, hepaticdecompensation, renal insufficiency, poorly controlled autoimmunedisease, severe cardiopulmonary disease, and active psychologicalproblems. [Davis G. L., Investigational Small-Molecule Agents for theTreatment of to Chronic Hepatitis C, in The Future of HCV: Smallmolecules in Development for Chronic Hepatitis C, Clinical Care OptionsLLC, 2007].

Briolant et al. [Antiviral Research 61 (2004) 111-117 teach that acombination of IFN-α2b and ribavirin has a subsynergistic anti-viraleffect on CHIKV and SFV.

Alternative therapies for the treatment of HCV related diseases havebeen developed. U.S. Pat. No. 6,849,254 discloses a combination therapyincluding the administration of interferon alfa and ribavirin for a timesufficient to lower HCV-RNA, in association with an antioxidant for atime sufficient to improve ribavirin-related hemolysis.

U.S. Pat. No. 7,115,578 discloses a combination therapy comprisingadministering a therapeutically effective amount of ribavirinderivatives and a therapeutically effective amount of interferon-alfa.U.S. Pat. No. 7,410,979 discloses a synergistically effectivecombination therapy of dihaloacetamide compounds and interferon orribavirin against HCV infection. U.S. Pat. No. 7,671,017 discloses theuse of cyclosporine and pegylated interferon for treating HCV.

Chloroquine is a well known lysosomotropic agent, currently attractingmany hopes in terms of antiviral therapy as well as in antitumoraleffect because of its pH-dependent inhibiting action on the degradationof cargo delivered to the lysosome, thus effectively disabling thisfinal step of the autophagy pathway.

Hydroxychloroquine (HCQ) is a chemical derivative of chloroquine (CQ)which features a hydroxyethyl group instead of an ethyl group.

HCQ has been classified as an effective anti-malarial medication, andhas shown efficacy in treating systemic lupus erythematosus as well asrheumatoid arthritis and Sjögren's Syndrome. While HCQ has been knownfor some time to increase lysosomal pH in antigen presenting cells, itsmechanism of action in inflammatory conditions has been only recentlyelucidated and involves blocking the activation of toll-like receptorsto on plasmacytoid dendritic cells (PDCs).

A direct comparison of the therapeutic effect of CQ and HCQ is quitedifficult but it has been suggested that hydroxychloroquine was one-halfto two-thirds as effective as chloroquine in treating rheumatologicdiseases and one-half as toxic [Scherbel A L et al., Cleve Clin Q, 1958,25:95]. Since chloroquine appears to be much more retinotoxic frequentuse of hydroxychloroquine is increasing [Rynes R. I., British Journal ofRheumatology, 1997; 36:799-805].

Chandramohan M. et al. [Indian J Pharm Sci 2006; 68:538-40] havereported the screening of chloroquine and hydroxychloroquine forpotential in vitro antiviral activity against HCV in Huh 5-2 cells, andshowed that chloroquine was able to reduce the viral RNA to below 7% andpromoted cell growth to more than 91% with respect to the untreatedcontrol at the concentration of 10.75 μM, and that hydroxychloroquinewas able to reduce the viral RNA to below 7% and promoted cell growth tomore than 81% with respect to the untreated control at the concentrationof 6.6 μM. Chandramohan M. et al. neither demonstrate that HCQ may beused in combination with an antiviral agent to treat HCV relateddiseases, nor characterize the effect of such a combination (antagonist,additive, or synergistic).

Freiberg et al. [Journal of General Virology (2010), 91, 765-772] haveevaluated the antiviral efficacy of chloroquine, individually and incombination with ribavirin, in the treatment of NiV and HeV infection inin vivo experiments, using a golden hamster model, and have reportedthat while both drugs exhibit a strong antiviral activity in inhibitingviral spread in vitro, they did not prove to be protective in the invivo model. Ribavirin delayed death from viral disease in NiV-infectedhamsters by approximately 5 days, but no significant effect inHeV-infected hamsters was observed. Chloroquine did not protect hamsterswhen administered either individually or in combination with ribavirin,the latter indicating the lack of a favorable drug-drug interaction.

Zuckerman et al. [BioDrugs 2001; 15(9), pp. 574-584] suggest that oraladministration of drugs such as HCQ, corticosteroids and otheranti-inflammatory agents can be combined with anti-viral therapy forcontrolling HCV-related arthritis.

Mizui et al. [J Gastroenterol. 2010 February; 45(2):195-203. Epub 2009Sep. 17] have later reported that treatment of cells transfected withHCV replicon with chloroquine suppressed the replication of the HCVreplicon in a dose-dependent manner. It was shown that a treatment withchloroquine, a known inhibitor of autophagy, and interferon-alfaenhanced the antiviral effect of the interferon, thereby preventingre-propagation of HCV replicon. Mizui et al. did not demonstrate anysynergistic or additive effect of the combination of CQ/interferon alfa,and did not relate to HCQ as a putative drug for HCV combinationtherapy.

A recent approach of gene expression profiling of JFH1 HCV infected Huh7cells showed that infection clearly modulates expression of host genesinvolved in several cellular processes such as ER stress response,apoptosis, p53 signaling, detoxification, intracellular lipidmetabolism, protein synthesis and degradation, post translationalprocesses or cytoskeleton organization.

The link between autophagy, a mechanism for cell survival in response tocellular stress role in cell death, and viral replication, including incases of HCV infection, is currently being investigated.

Autophagy, a cellular pathway leading to components self-degradation, isknown to be activated in response to stress, including ER stressinitiated after viral infection. Although autophagy provides protectionagainst various infections, and has been described as a component of theinnate immune response, several bacteria and viruses, including HCV,have developed strategies to subvert autophagic processes to facilitatetheir own replication [Schmid & Munz, Immunity 2007, 27:11-21; Wileman,Science 2006, 312:875-878]. It was found that silencingautophagy-related genes significantly blunts the replication of HCV[Mizui et al., 2010 supra] and decreases the production of infectiousHCV particles. It has been suggested that induction of autophagy by HCVimpairs the innate immune response, and disruption of autophagy inHCV-infected hepatocytes activates the interferon signaling pathway andenhances the innate immune response [Shrivastava et al., Hepatology2011, 53:406-414].

HCV infection, in vitro and in vivo, induces ER stress and triggersautophagy through the induction of unfolded protein response (UPR)including the downstream IRE1, ATF6, and EIF2AK3/PERK signaling pathways[Sir et al., Hepatology 2008, 48:1054-1061]. However, HCV-inducedautophagic process was thought to be incomplete since it does not leadto protein degradation [Sir et al., Hepatology 2008, 48:1054-1061].

Ke P Y. et al. have reported that a completed autophagic process totallysuppresses innate antiviral immunity, through a blockade of theendogenous IFN response, allowing HCV RNA replication.

Although the main autophagy proteins seem to be proviral factorsrequired for the translation of incoming HCV RNA, and thereby for theinitiation of HCV replication, it has been suggested that autophagy isnot required once infection is established [see, for example, Dreux etal., PNAS 2009, 106:14046-14051].

However, neither the host molecular mechanisms involved in response toHCV infection, nor the molecular basis of the antiviral activity of CQand its interplay with autophagy have been clearly elucidatedheretofore.

Recently, it has been suggested that autophagy is involved also incancer therapy, as being induced by chemotherapy such as DNA-damagingchemotherapy, radiation therapy, and molecularly targeted therapies, andthat chloroquine derivatives such as HCQ may be used to prevent thechemotherapy-induced autophagy [Ravi K. Amaravadi, J Clin Invest. 2008;118(12):3837-3840].

Additional background art includes Jackson et al. [PLoS Biol 2005,3:e156], Wong et al. [J Virol 2008, 82:9143-9153], Khakpoor et al. [JGen Virol 2009, 90:1093-1103], Lee et al. [Virology 2008, 378:240-248],Prentice et al. [J Bio Chem 2004, 279:10136-10141], and Kroemer et al.[Nat Rev Mol Cell Biol 2008, 9:1004-1010].

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the invention, there isprovided a method of treating an HCV infection, the method comprising:

(a) identifying an HCV-infected subject non-responsive to an anti-HCVtherapy; and

(b) administering to the HCV-infected subject a therapeuticallyeffective amount of hydroxychloroquine or a pharmaceutically acceptablesalt thereof, thereby treating the HCV infection.

According to an aspect of some embodiments of the present invention,there is provided a method of treating a hepatitis C virus (HCV) relateddisease in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount ofhydroxychloroquine or a pharmaceutically acceptable salt thereof, thetherapeutically effective amount being an amount sufficient to inhibitHCV-induced autophagy in the subject, thereby treating the HCV relateddisease.

According to an aspect of some embodiments of the present invention,there is provided hydroxychloroquine identified for use in the treatmentof a hepatitis C virus (HCV) related disease in an amount sufficient toinhibit HCV-induced autophagy.

According to an aspect of some embodiments of the present invention,there is provided a use of a hydroxychloroquine in the manufacture of amedicament for use in the treatment of a hepatitis C virus (HCV) relateddisease, the treatment comprising administering to a subject in needthereof hydroxychloroquine in an amount sufficient to inhibitHCV-induced autophagy.

According to an aspect of some embodiments of the present invention,there is provided a method of treating an hepatitis C virus (HCV)related disease in a subject in need thereof, the method comprisingco-administering to the subject a therapeutically effective amount ofhydroxychloroquine or a pharmaceutically acceptable salt thereof and atherapeutically effective amount of an antiviral agent, wherein theanti-viral agent does not inhibit HCV-induced autophagy, therebytreating the HCV infection.

According to an aspect of some embodiments of the present invention,there is provided hydroxychloroquine for use in the treatment of ahepatitis C virus (HCV) related disease in combination with an antiviralagent that does not inhibit HCV-induced autophagy.

According to an aspect of some embodiments of the present invention,there is provided a use of hydroxychloroquine in the manufacture of amedicament for use in the treatment of a hepatitis C virus (HCV) relateddisease in combination with an anti-viral agent that does not inhibitHCV-induced autophagy.

According to an aspect of some embodiments of the present invention,there is to provided a method of treating an HCV related disease causedby a hepatitis C virus (HCV) genotype resistant to an antiviral agent ina subject in need thereof, the method comprising co-administering to thesubject a therapeutically effective amount of hydroxychloroquine or apharmaceutically acceptable salt thereof and a therapeutically effectiveamount of the antiviral agent, thereby treating the HCV related disease.

According to an aspect of some embodiments of the present invention,there is provided hydroxychloroquine, for use in combination with anantiviral agent in the treatment of an HCV-related disease caused by ahepatitis C virus (HCV) genotype resistant to the antiviral agent.

According to an aspect of some embodiments of the present invention,there is provided a use of hydroxychloroquine in the manufacture of amedicament for use in combination with an antiviral agent in thetreatment of an HCV-related disease caused by a hepatitis C virus (HCV)genotype resistant to the antiviral agent.

According to an aspect of some embodiments of the present invention,there is provided a method of treating a hepatitis C virus (HCV) relateddisease in a subject in need thereof, the method comprisingco-administering to the subject a therapeutically effective amount ofhydroxychloroquine or a pharmaceutically acceptable salt thereof incombination with a therapeutically effective amount of an antiviralagent, wherein the therapeutically effective amount ofhydroxychloroquine and the therapeutically effective amount of theantiviral agent are selected such that hydroxychloroquine and theanti-viral agent act in synergy, thereby treating the HCV relateddisease.

According to an aspect of some embodiments of the present invention,there is provided a method of treating a hepatitis C virus (HCV) relateddisease in a subject in need thereof, the method comprisingco-administering to the subject from 400 to 2000 mg per day ofhydroxychloroquine or a pharmaceutically acceptable salt thereof incombination with from 50 to 250 μg per week of an interferon, whereinthe hydroxychloroquine and the interferon act in synergy, therebytreating the HCV related disease.

According to an aspect of some embodiments of the present invention,there is provided a method of treating a hepatitis C virus (HCV) relateddisease in a subject in need thereof, the method comprisingco-administering to the subject from 400 to 2000 mg per day ofhydroxychloroquine or a pharmaceutically acceptable salt thereof incombination with a therapeutically effective amount of a viral proteaseinhibitor, wherein the hydroxychloroquine and the viral proteaseinhibitor act in synergy, thereby treating the HCV related disease.

According to an aspect of some embodiments of the present invention,there is provided a method of treating a hepatitis C virus (HCV) relateddisease in a subject in need thereof, the method comprisingco-administering to the subject from 400 to 2000 mg per day ofhydroxychloroquine or a pharmaceutically acceptable salt thereof incombination with a therapeutically effective amount of a viralpolymerase inhibitor, wherein the hydroxychloroquine and the viralpolymerase inhibitor act in synergy, thereby treating the HCV relateddisease.

According to an aspect of some embodiments of the present invention,there is provided hydroxychloroquine, identified for use in combinationwith an antiviral agent in the treatment of a hepatitis C virus (HCV)related disease, wherein the hydroxychloroquine and the antiviral agentact in synergy.

According to an aspect of some embodiments of the present invention,there is provided a use of hydroxychloroquine in the manufacture of amedicament identified for use in combination with an antiviral agent inthe treatment of a hepatitis C virus (HCV) related disease, wherein thehydroxychloroquine and the antiviral agent act in synergy.

According to an aspect of some embodiments of the present invention,there is provided a pharmaceutical composition comprisinghydroxychloroquine or a pharmaceutically acceptable salt thereof, anantiviral agent, and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present invention,there is provided a pharmaceutical composition unit dosage formcomprising hydroxychloroquine or a pharmaceutically acceptable saltthereof, an antiviral agent, and a pharmaceutically acceptable carrier.

According to some embodiments of the invention, the antiviral agent isribavirin.

According to some embodiments of the invention, the pharmaceuticalcomposition or pharmaceutical composition unit dosage form is identifiedfor use in the to treatment of a hepatitis C virus (HCV) infection in anHCV-infected subject non-responsive to an anti-HCV therapy.

According to some embodiments of the invention, the abovementionedanti-HCV therapy comprises a treatment with PEGylated interferon α-2a orPEGylated interferon α-2b, in combination with ribavirin.

According to some embodiments of the invention, the HCV-infected subjectis lacking a sustained virological response (SVR).

According to some embodiments of the invention, the pharmaceuticallyacceptable salt is hydroxychloroquine sulfate.

According to some embodiments of the invention, the unit dosage formcomprises an amount of hydroxychloroquine sulfate in a range of fromabout 400 to about 600 mg.

According to some embodiments of the invention, the therapeuticallyeffective amount of hydroxychloroquine or a pharmaceutically acceptablesalt thereof is in a range of from about 400 to about 2000 mg per day.

According to some embodiments of the invention, the therapeuticallyeffective amount of hydroxychloroquine or a pharmaceutically acceptablesalt thereof is in a range of from about 500 to about 1000 mg per day.

According to some embodiments of the invention, the method furthercomprises administering to the HCV-infected subject a therapeuticallyeffective amount of at least one antiviral agent.

According to some embodiments of the invention, the method furthercomprises administering to the HCV-infected subject a therapeuticallyeffective amount of an interferon.

According to some embodiments of the invention, the interferon isPEGylated interferon α-2a.

According to some embodiments of the invention, the HCV-inducedautophagy is characterized by an increase in a level of a proteinselected from the group consisting of ULK1, AMBRA1, ATG2A, GABARAPL1,FOX03, SQSTM1, PIK3C3 and MAP1LC3B.

According to some embodiments of the invention, the disease is caused byan anti-viral resistant HCV genotype.

According to some embodiments of the invention, the HCV is selected fromthe group consisting of genotype 1 HCV and genotype 4 HCV.

According to some embodiments of the invention, the HCV-infected subjectis infected by genotype 1 HCV.

According to some embodiments of the invention, the HCV-infected subjectis infected by genotype 1b HCV.

According to some embodiments of the invention, the method furthercomprises co-administering to the subject a therapeutically effectiveamount of an antiviral agent.

According to some embodiments of the invention, the antiviral agent isselected from the group consisting of ribavirin, viramidine, aninterferon, a viral protease inhibitor, an NS4A inhibitor, an NS5Ainhibitor, a viral polymerase inhibitor, a cyclophilin inhibitor, ahelicase inhibitor, a glycosylation inhibitor, and an antiphospholipidantibody, and any combination thereof.

According to some embodiments of the invention, the antiviral agent isselected from the group consisting of ribavirin, a viral proteaseinhibitor, an NS4A inhibitor, an NS5A inhibitor, and a viral polymeraseinhibitor, and any combination thereof.

According to some embodiments of the invention, the antiviral agent isselected from the group consisting of an interferon-α, ribavirin,viramidine, boceprevir, telaprevir, NM-107, valopicitabine, andalisporivir, and any combination thereof.

According to some embodiments of the invention, the anti-viral agentdoes not inhibit HCV-induced autophagy.

According to some embodiments of the invention, the anti-viral agent isselected from the group consisting of ribavirin, viramidine, boceprevir,telaprevir, NM-107, valopicitabine, and alisporivir, and any combinationthereof.

According to some embodiments of the invention, the method furthercomprises co-administering to the subject a therapeutically effectiveamount of an interferon-α.

According to some embodiments of the invention, the amount ofhydroxychloroquine sufficient to inhibit HCV-induced autophagy is in arange of from 400 to 2000 mg per day.

According to some embodiments of the invention, the treatment furthercomprises co-administering a therapeutically effective amount of anantiviral agent.

According to some embodiments of the invention, the therapeuticallyeffective amount of hydroxychloroquine is sufficient to inhibitHCV-induced autophagy in the subject.

According to some embodiments of the invention, the therapeuticallyeffective amount of hydroxychloroquine is in a range of from 500 to 1000mg per day.

According to some embodiments of the invention, the method furthercomprises co-administering to the subject a therapeutically effectiveamount of an additional anti-viral agent.

According to some embodiments of the invention, the additional antiviralagent is selected from the group consisting of an interferon, a viralprotease inhibitor, an NS4A inhibitor, an NS5A inhibitor, a viralpolymerase inhibitor, a cyclophilin inhibitor, a helicase inhibitor, aglycosylation inhibitor, and an antiphospholipid antibody.

According to some embodiments of the invention, the therapeuticallyeffective amount of ribavirin is in a range of from 50 to 1200 mg perday.

According to some embodiments of the invention, the disease is caused byan HCV genotype that is resistant to the antiviral agent.

According to some embodiments of the invention, the method furthercomprises co-administering to the subject an additional anti-viralagent.

According to some embodiments of the invention, the additional antiviralagent is selected from the group consisting of an interferon, a viralprotease inhibitor, an NS4A inhibitor, an NS5A inhibitor, a viralpolymerase inhibitor, a cyclophilin inhibitor, a helicase inhibitor, aglycosylation inhibitor, and an antiphospholipid antibody, and anycombination thereof.

According to some embodiments of the invention, the treatment comprisesadministration of hydroxychloroquine in an amount sufficient to inhibitHCV-induced autophagy.

According to some embodiments of the invention, the treatment comprisesadministration of from 400 to 2000 mg per day of hydroxychloroquine.

According to some embodiments of the invention, the treatment comprisesadministration of from 500 to 1000 mg per day of hydroxychloroquine.

According to some embodiments of the invention, the antiviral agent is aviral protease inhibitor.

According to some embodiments of the invention, the viral proteaseinhibitor is boceprevir.

According to some embodiments of the invention, the antiviral agent is aviral polymerase inhibitor.

According to some embodiments of the invention, the viral polymeraseinhibitor is selected from the group consisting of NM-107 andvalopicitabine.

According to some embodiments of the invention, the antiviral agent isan interferon.

According to some embodiments of the invention, the interferon is aninterferon-α.

According to some embodiments of the invention, the interferon-α is aPEGylated interferon-α.

According to some embodiments of the invention, the therapeuticallyeffective amount of an interferon is in a range of from 50 to 250 μg perweek.

According to some embodiments of the invention, the treatment comprisesadministration of hydroxychloroquine at a dosage of from 400 to 2000 mgper day.

According to some embodiments of the invention, the treatment comprisesadministration of the interferon at a dosage in a range of from 50 to250 μg per week.

According to some embodiments of the invention, the treatment furthercomprises co-administration of a therapeutically effective amount of anadditional antiviral agent.

According to some embodiments of the invention, the composition isidentified for use in the treatment of a hepatitis C virus (HCV) relateddisease.

According to some embodiments of the invention, the composition isformulated for oral administration.

According to some embodiments of the invention, the composition is in asolid form.

According to some embodiments of the invention, the composition is aunit dosage form of the composition.

According to some embodiments of the invention, the unit dosage form isidentified for use in the treatment of a hepatitis C virus (HCV) relateddisease.

According to some embodiments of the invention, the unit dosage form isformulated for oral administration.

According to some embodiments of the invention, the unit dosage form isin a solid form.

According to some embodiments of the invention, the unit dosage formcomprises an amount of hydroxychloroquine sufficient to inhibitHCV-induced autophagy.

According to some embodiments of the invention, the amount ofhydroxychloroquine is sufficient to sensitize hepatitis C virus (HCV) tothe antiviral agent.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a drawing showing the structure of the HCV RNA replicon ofcell line Huh 7;

FIG. 2 is a graph showing the level of HCV RNA in replicon cellsfollowing treatment for 72 hours with a combination of 0, 0.22, 0.66, 2,6 or 18 μM hydroxychloroquine with 0, 0.41, 1.23, 3.7, 11.1, 33.3, 100or 300 IU/ml interferon-α (IFNαA);

FIGS. 3A and 3B are graphs showing the difference between a measuredantiviral effect of a combination of 0, 0.22, 0.66, 2, 6 or 18 μMhydroxychloroquine with 0, 0.41, 1.23, 3.7, 11.1, 33.3, 100 or 300 IU/mlinterferon-α (IFNa) and the theoretical antiviral effect expectedaccording to a Prichard-Shipman model of an additive effect (positivevalues indicate synergy, negative values indicate antagonism); the datais presented in 3-dimensional (FIG. 3A) and 2-dimensional (FIG. 3B)schemes;

FIG. 4 presents an isobologram showing the concentrations ofinterferon-α(IFNa) and hydroxychloroquine which provide 50% (ED50), 75%(ED75) and 90% (ED90) antiviral inhibition, as determined experimentally(data points) and as calculated by a theoretical model for an additiveeffect (dotted lines);

FIG. 5 is a graph showing the viability of HCV replicon cells followingtreatment for 72 hours with a combination of 0, 0.22, 0.66, 2, 6 or 18μM hydroxychloroquine with 0, 0.41, 1.23, 3.7, 11.1, 33.3, 100 or 300IU/ml interferon-α (IFNαA);

FIG. 6 is a graph showing the difference between a measured antiviraleffect of a combination of 0, 0.66, 2, 6 or 18 μM chloroquine with 0,0.41, 1.23, 3.7, 11.1, 33.3, or 100 IU/ml interferon-α (IFN-a A) and thetheoretical antiviral effect expected according to a Prichard-Shipmanmodel of an additive effect (positive values indicate synergy, negativevalues indicate antagonism);

FIG. 7 is a bar graph showing the level of HCV RNA in replicon cellsfollowing treatment for 72 hours with a combination of 0, 0.5, 1 or 10μM hydroxychloroquine (HCQ), as determined by quantification of areplicon-borne neomycin gene product (NPTII);

FIG. 8 is an image of a Western blot showing levels of HCV core proteinand HCV NS5A protein following treatment of Huh7 replicon cells for 48hours with 0.5 or 1 μM hydroxychloroquine (HCQ) or without treatment(NT) (α-actin served as a loading control);

FIG. 9A is a bar graph showing the percentage of host genes (out of atotal of to 10238 significantly expressed genes) which are modulated(up-regulated or down-regulated) in Huh7 cells 6, 24 or 48 hours afterHCV infection with JFH/CsN6A4 viral particles;

FIG. 9B shows the expression profiles of host genes modulated in Huh7cells 6, 24 or 48 hours after HCV infection with JFH/CsN6A4 viralparticles, or 12 or 48 hours after HCV infection with treatment with HCQ(yellow bar) or CQ (blue bar); red indicates up-regulation, greenindicates down-regulation, and black indicates no modulation;

FIG. 9C is a bar graph showing the modulation of 118 genes, out of the1736 modulated (induced or repressed) 48 hours after HCV infection withJFH/CsN6A4 viral particles, which were overexpressed or down-regulated(at least a 2-fold modulation in expression) 48 hours after HCQ and CQtreatment (each bar represents the proportion of genes modulated bytreatment among the HCV-induced genes or among the HCV-repressed genes;white indicates the proportion of genes down-regulated by treatment,black indicates the proportion of genes up-regulated by treatment);

FIG. 10 shows a set of 57 genes involved in the gene regulatory network,and HCQ (blue bar) and CQ (yellow bar) repression in Huh7 cells 48 hoursafter infection with JFH/CsN6A4 viral particles (expression 6, 24 and 48hours after infection, without HCQ or CQ treatment is shown forcomparison; red bar indicates expression in infected cells treated withinterferon-α);

FIG. 11 is a gene network showing HCQ inhibition of HCV-inducedpathways;

FIG. 12 shows expression of exemplary modulated genes in infected cells(Hi/H), and in infected cells treated with HCQ (HiCQd/Hi), CQ (HiCQ/Hi)or interferon-α (HiIFN/Hi), as determined by microarray analysis,SYBRGreen qRT-PCR, and Nanostring techniques;

FIGS. 13A and 13B are graphs showing the difference between a measuredantiviral effect of a combination of 0, 0.22, 0.66, 2, 6 or 18 μMhydroxychloroquine with 0, 0.11, 0.33, 1, 3.3 or 10 μM NM-107 and thetheoretical antiviral effect expected according to a Prichard-Shipmanmodel of an additive effect (positive values indicate synergy, negativevalues indicate antagonism); the data is presented in 3-dimensional(FIG. 13A) and 2-dimensional (FIG. 13B) schemes; and

FIGS. 14A and 14B are graphs showing the difference between a measuredto antiviral effect of a combination of 0, 0.22, 0.66, 2, 6 or 18 μMhydroxychloroquine with 0, 0.041, 0.123, 0.37, 1.1, 3.3 or 10 μMboceprevir and the theoretical antiviral effect expected according to aPrichard-Shipman model of an additive effect (positive values indicatesynergy, negative values indicate antagonism); the data is presented in3-dimensional (FIG. 14A) and 2-dimensional (FIG. 14B) schemes.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to therapyand, more particularly, but not exclusively, to methods and compositionsfor the treatment of Hepatitis C Virus (HCV) related diseases such aschronic HCV infection.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

In a search for a novel methodology for treating HCV related diseases,and chronic HCV infection in particular, which would obviate thenon-responsiveness and/or non-tolerance associated with the currentmethodologies, the present inventors have surprisingly uncovered thathydroxychloroquine (HCQ) can be used as an agent for treatingHCV-related diseases, either alone or in combination with one or moreanti-viral agents.

As demonstrated in the Examples section that follows, the presentinventors have shown that HCQ acts in synergy with anti-viral agentssuch as interferon-α (IFNα), boceprevir and NM-107, thus providing foran improved therapeutic effect of these agents and/or allowing usinglower doses of the anti-viral agent(s).

Thus, it is demonstrated herein that exemplary combinations of HCQ andIFNα exhibit a synergistic effect, as determined by various theoreticalmodels (FIGS. 2-4 and Table 1). In addition, it is demonstrated thatexemplary combinations of HCQ and IFNα exhibit an antiviral effect evenat concentrations of HCQ and/or IFNα which do not exhibit an antiviraleffect when administered alone (FIG. 2). Exemplary combinations whichexhibited the aforementioned synergistic effects were non-toxic (FIG.5). Furthermore, it was demonstrated that chloroquine does not exhibitthe same synergistic effects as HCQ (FIG. 6), indicating that theaforementioned advantageous properties of HCQ are not a general propertyof chloroquine-related compounds.

Further it is demonstrated that exemplary combinations of HCQ andantiviral agents such as boceprevir (a protease inhibitor) and NM-107(HCV polymerase inhibitor) exhibit a synergistic effect.

It has been shown that synergy was best achieved when cells were treatedwith HCQ at a concentration of about 6 μM (See, for example, FIGS.3A-3B, and 13A-14B). This dose can be translated, using recognizedconversion factors, into a dose of about 910 mg for an average humanweighing 70 Kg.

While exploring the mechanism of action of HCQ, the present inventorshave demonstrated that HCQ treatment effectively reduces the expressionlevel of several HCV-induced NF-κB signaling, ER stress, autophagy andp53 signaling pathways. In addition, it has been demonstrated that IFNand HCQ have a similar repressive effect on these HCV-induced pathways.Without being bound by any particular theory, these findings suggestthat the HCQ inhibitory modulations of the HCV-activated biologicalpathways reflect a consequence of an upstream antiviral eventefficiently triggering the HCV eradication.

Preliminary analysis demonstrated that HCQ treatment leads concomitantlyto 1) a dose-dependent decrease in the number of HCV infected cells(FIG. 7), 2) a dose-dependent decrease of levels of HCV NS5A and coreprotein (FIG. 8), and 3) a decrease in the level of mRNA for severalNF-κB related genes, particularly 24 hours after treatment initiation(Tables 2-3). The HCQ-induced reduction of the NS5A and the HCV coreprotein levels is in agreement with the decrease observed in expressionof several NF-κB related genes (e.g. RELB, NFKB2, CXCL5 and CYR61).

Interestingly, it has been previously reported that the NS5A and the HCVcore protein can lead to the activation of the NF-κB through distinctsignaling pathways: Ca2+ disturbance resulting from an endoplasmicreticulum (ER) stress or through TNF signaling, respectively. Othersstudies have demonstrated several lines of evidence of the NF-κB pathwayactivation in HCV infection both in vitro and in vivo. Thus,constitutive activation of NF-κB by HCV might have implications inchronic liver disease including hepatocellular carcinoma associated withHCV infection.

Further gene expression analysis performed on the JFH1 HCVcc model, anaccurate model of physiological HCV infection, revealed that HCVinfection increases the expression of several NF-κB related genes, asexpected. Moreover, in agreement with previous findings using a HCVreplicon model, it was demonstrated that HCQ treatment severely reducesexpression of these HCV-induced NF-κB related genes, such as RELB,CYR61, CXCL5 (previously found repressed by HCQ treatment of HCVreplicon cells), BCL3, CY1PA1, NFKBIA,B,E and NKIRAS1 (FIGS. 9B and 10).Altogether, these findings demonstrate that HCQ counteracts theHCV-induced increase of expression levels of NF-κB related genes.Furthermore, comparison with expression ratios obtained followingchloroquine treatment showed that HCQ is more effective than chloroquineat counteracting the HCV-induced increase.

In addition, global gene expression profiling demonstrates that HCVinfection induced several others pathways, including ER stress/UPR(unfolded protein response) and the p53 signaling apoptotic cell death,and also cell cycle and lipid metabolism pathways (FIG. 9B).Interestingly, similarly to NF-κB, for all these processes, HCQtreatment modulated the process in the opposite manner to which HCVinfection modulated the process, particularly 48 hours post-infectionand treatment (FIGS. 9A-9C).

118 genes were determined to undergo at least a twofold change underboth of the following conditions: HCV infection, and HCQ andhydroxychloroquine treatment, after 48 hours. It was demonstrated thatmost of these genes are genes whose expression is up-regulated by HCVand down-regulated by HCQ treatment (FIG. 9C and Table 5). Resultsobtained using both HCV replicon (Table 2) and HCVcc (Tables 4-5) modelswere in agreement, indicating that HCQ acts primarily as atranscriptional repressor, with considerably more down-regulation beingobserved than up-regulation.

Of the abovementioned 118 genes, 57 genes were found to be particularlysignificant, in that they were differentially expressed in theaforementioned two study conditions, or found to be functionally relatedto significant genes according to the text-mining based analysisperformed using PredictSearch™ (Table 6).

Based on the obtained results, a gene regulatory network was constructedshowing the inhibitory effect of HCQ treatment on JFH1-induced Huh7 geneexpression modulations (FIG. 11). As shown in FIG. 11, a significantresult of HCQ treatment to is the inhibition of the autophagy pathway(as a result of inhibition of upstream NF-κB, p53 and ER stress/UPRsignaling), which interferes with HCV replication.

The above-described findings suggest that treatment of an HCV relateddisease such as an HCV infection can be beneficially effected whileutilizing HCQ in an amount sufficient to inhibit HCV-induced autophagy.

Since autophagy is a pathway which is unrelated to the HCV genotype,these findings further indicate a role of HCQ in the treatment ofresistant HCV genotypes, which are otherwise characterized by lowresponsiveness to current therapy. Moreover, these findings suggest arole for HCQ in a combined HCV treatment with anti-viral agents, via anadditive and even synergistic effect of the HCQ and the anti-viralagent(s) and/or via sensitizing the HCV to the antiviral agent,presumably by inhibiting HCV-induced pathways such as HCV-inducedautophagy.

It was further shown in the Examples that co-administration of HCQ iscapable of potentiating the activity of anti-HCV agents such asPEGylated interferon and ribavirin in the treatment of HCV-infectedpatients which failed to respond to a standard of care treatment.

It is further noted that HCQ is known as a less toxic derivative ofchloroquine. Thus, higher doses of HCQ can be used, compared tochloroquine.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a hepatitis C virus (HCV) relateddisease in a subject in need thereof. The method, according to thisaspect of the present invention, is effected by administering to thesubject a therapeutically effective amount of hydroxychloroquine (HCQ)or a pharmaceutically acceptable salt thereof.

In some embodiments, the therapeutically effective amount of HCQ is anamount sufficient to inhibit HCV-induced autophagy in the subject,thereby treating the HCV related disease.

As used herein throughout, the term “hepatitis C virus”, abbreviatedHCV, describes an enveloped, positive-sense single-stranded RNA virus ofthe family Flaviviridae, which is the cause of hepatitis C in humans,and encompasses all genotypes of the virus, unless otherwise indicated.

Currently known HCV genotypes include genotypes 1, 2, 3, 4, 5 and 6, ofwhich genotypes 1 and 4 are relatively non-responsive to existingtreatments (e.g., interferon to and ribavirin), whereas genotypes 2, 3,5 and 6 are more responsive to treatment.

In some embodiments, the HCV related disease is an infection caused byHCV, including all genotypes of HCV, as defined herein.

In some embodiments, the infection caused by HCV is an acute infection,encompassing any acute phase of an HCV infection (e.g., the first 6months after infection), also termed herein and in the art “acute HCVinfection”.

An “acute HCV infection” thus relates to an infection which has beeneliminated, namely, to cases where viral replication has been eliminatedand the virus is eradicated.

If viral replication is not successfully inhibited within the acutephase, the HCV infection is considered a chronic infection.

In some embodiments, the infection caused by HCV is a chronic infection,also termed herein and in the art “chronic HCV infection”.

In some embodiments, treating an HCV infection encompasses treating anacute HCV infection, and preventing an acute HCV infection from beingconsidered as a chronic HCV infection.

As used herein throughout, the phrase “HCV related disease” alsoencompasses any disease or disorder associated with an HCV infection,including symptoms associated with an acute HCV infection, such asdecreased appetite, fatigue, abdominal pain, jaundice, itching andflu-like symptoms, as well as symptoms associated with a chronic HCVinfection, such as fatigue, flu-like symptoms, joint pains, arthritis,polyarthralgia, cutaneous leukocytoclastic vasculitis, neuropathy,itching, sleep disturbances, appetite changes, nausea, depression, livercirrhosis, ascites, a tendency towards bruising and/or bleeding,varices, jaundice, hepatic encephalopathy, porphyria cutanea cardia,cryoglobulinemia, glomerulonephritis (e.g., membranoproliferativeglomerulonephritis), thrombocytopenia, lichen planus, diabetes mellitus,and lymphoproliferative disorders.

Some types of HCV related disease are inflammatory conditions (e.g.,arthritis) which may be treated by a relatively simple anti-inflammatorytherapy. However, other types of HCV related disease may be moredifficult to treat.

In some embodiments, the phrase “HCV related disease” refers to an HCVdisease other than such inflammatory conditions (e.g., other thanarthritis).

In addition, disease caused by an HCV genotype resistant to an antiviralagent (antiviral-resistant) may be particularly difficult to treatsuccessfully.

Currently known resistant HCV genotypes include genotype 1 HCV andgenotype 4 HCV. These genotypes are known in the art to exhibitresistance to antiviral agents commonly used to treat HCV relateddisease.

In some embodiments, the HCV related disease is caused by anantiviral-resistant HCV genotype.

Resistance can be inherent to an organism or acquired (e.g., as a resultof exposure to an antiviral agent resulting in selection for a mutantgenotype resistant to the agent). When resistance is acquired uponexposure to an antiviral agent, the resistance may be specific to thatantiviral agent (and in some cases highly similar antiviral agents), orthe acquired resistance may to a variety of antiviral agents, includingantiviral agents to which the organism was not exposed.

As used herein, a “subject” describes any mammal afflicted, or suspectedas being afflicted, by an HCV related disease as described herein,and/or to whom the treatment methods described herein are desired,including human, bovine, equine, canine, murine and feline subjects. Insome embodiments, the subject is a human.

In some embodiments, “a subject in need thereof” is a subject diagnosedas having an HCV-related disease. Determining an HCV-related disease canbe made by blood tests for detecting antibodies to HCV, and molecularnucleic acid tests for detecting the presence of HCV (e.g., polymerase,chain reaction, transcription mediated amplification and/or branched DNAmethods). Optionally, both antibody and nucleic acid tests are used, inorder to confirm that an HCV infection is present. The particularHCV-related disease can be determined by a physician using standardmethods (e.g., physical examination, liver function tests), depending onwhich symptoms are present in a subject.

In some embodiments “a subject in need thereof” is a subject who isafflicted by an HCV-related disease, such as chronic HCV infection, andwho was treated with an anti-viral agent or a combination of anti-viralagents, but was identified as non-responsive to the treatment or asnon-tolerant to the treatment.

In some embodiments, the subject is an HCV-infected subject (i.e., asubject who is afflicted by an HCV infection), and who is identified asbeing non-responsive to to an anti-HCV therapy. In some embodiments, thesubject is identified as being non-responsive to an anti-HCV therapywhich is a treatment with PEGylated interferon-α-2a or PEGylatedinterferon-α-2b, in combination with ribavirin.

As used herein, the term “non-responsive” refers to a failure of anantiviral therapy used in the art against HCV (e.g., a treatment withPEGylated interferon-α-2a or PEGylated interferon-α-2b, in combinationwith ribavirin), and optionally a failure of two such antiviraltherapies, to abrogate, substantially inhibit, slow or reverse theprogression of an HCV-related disease, or substantially amelioratingclinical symptoms of an HCV-related disease.

In some embodiments, an HCV-infected subject is identified asnon-responsive to an anti-HCV therapy, when the subject is lacking asustained virological response (SVR), as determined in the art, to theanti-HCV therapy.

In some embodiments, non-responsiveness to treatment is a result of anHCV (e.g., an HCV genotype) which is resistant to the therapy.Alternatively or additionally, non-responsiveness is due to the subject(e.g., physiology of the subject, poor compliance by the subject). Insome embodiments, the reason(s) for non-responsiveness are not known.

In some embodiments, the HCV-infected subject is infected by genotype 1HCV. In exemplary embodiments, the HCV-infected subject is infected bygenotype 1b HCV.

As used herein, the term “non-tolerant” refers to the development of oneor more adverse effects in a treated subject, which are judged by aphysician to be due to the treatment, wherein the adverse effects aresufficiently severe such as to require ending or altering the treatment(e.g., by reducing the dosage of the anti-viral therapy and/or byreplacing the anti-viral therapy).

It is to be appreciated that a subject may be non-responsive andnon-tolerant to treatment. Thus, for example, a subject may benon-tolerant of relatively high dosages of an antiviral agent (e.g.,dosages of PEGylated interferon-α and/or ribavirin) used in the art,while also being non-responsive to relatively low dosages of anantiviral agent used in the art, such that the subject is non-tolerantand/or non-responsive to all possible dosages of the antiviral agent.

As used herein and is known in the art, the term “autophagy” describes aprocess in cell biology (also known in the art as “autophagocytosis”)involving degradation of a cell's own components via the lysosomalmachinery.

The phrase “HCV-induced autophagy” describes autophagy in a cell whichis due to infection of the cell by HCV.

As used herein, the phrases “an amount sufficient to inhibit HCV-inducedautophagy” and “an amount that inhibits HCV-induced autophagy” are usedinterchangeably, and encompass an amount of HCQ or of a salt thereofwhich reduces HCV-induced autophagy by at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or even which completely abolishes HCV-induced autophagy(i.e., restores levels of autophagy to normal levels). Any integerbetween 10% and 100% is contemplated in this regard.

HCV-induced autophagy, as well as inhibition of HCV-induced autophagy,can be determined, for example, by identifying autophagy or anautophagy-related process which occurs in infected cells, but not incorresponding non-infected cells.

In some embodiments, HCV-induced autophagy is characterized by anincrease in a level of one or more proteins associated with autophagy.Optionally, the increase is characterized by an increase of at least100%, in expression of the one or more proteins, as determined usingstandard techniques (e.g., microarray analysis, RT-PCR, and/orNanoString™ gene expression analysis). Exemplary proteins associatedwith HCV-induced autophagy include, but are not limited to, ULK1,AMBRA1, ATG2A, GABARAPL1, FOX03, SQSTM1, PIK3C3 and MAP1LC3B, which werefound to be upregulated in HCV-infected cells.

In some embodiments, HCV-induced autophagy is characterized by anincrease (e.g., of at least 100%) of the levels of at least 1,optionally at least 2, optionally at least 3, optionally at least 4,optionally at least 5, optionally at least 6, optionally at least 7, andoptionally all 8, of the aforementioned proteins.

In some embodiments, inhibition of HCV-induced autophagy (e.g.,inhibition of between 10% and 100%) is characterized by a reduction inan increase (e.g., of at least 100%) of the levels of at least 1,optionally at least 2, optionally at least 3, optionally at least 4,optionally at least 5, optionally at least 6, optionally at least 7, andoptionally all 8, of the aforementioned proteins. Thus, for example, atreatment-induced reduction by to 90% of an HCV-induced 100% increase ina protein level results in a 10% increase relative to a level innon-infected and non-treated cells.

In some embodiments, autophagy is determined by measuring thearteriovenous amino acid exchange rate in peripheral tissues, asdescribed in Klionsky et al. [Autophagy 2008, 4:151-175], which isincorporated by reference as if fully set forth herein.

As used herein, the term “hydroxychloroquine” includes the racemichydroxychloroquine, which is2-[[4-[(7-chloro-4-quinolinyl)amino]pentyl]ethylamino]ethanol asdisclosed in U.S. Pat. No. 2,546,658, or any of the single enantiomers“(S)-(+) hydroxychloroquine” or “(R)-(−) hydroxychloroquine” asdisclosed in U.S. Pat. No. 5,314,894. This term may relate either to thefree form of hydroxychloroquine or to any pharmaceutically acceptablesalt thereof, such as hydroxychloroquine sulfate.

Herein, the term “pharmaceutically acceptable salt” refers to a chargedspecies of the parent compound and its counter ion, which is typicallyused to modify the solubility characteristics of the parent compoundand/or to reduce any significant irritation to an organism by the parentcompound, while not abrogating the biological activity and properties ofthe administered compound. Examples, without limitation, ofpharmaceutically acceptable salts include salts comprising an anion suchas a carboxylate or sulfate anion, and/or a cation such as, but notlimited to, ammonium, sodium, potassium and the like. Suitable salts aredescribed in, e.g., Birge et al. [J Pharm Sci 1977, 66:1-19]. An exampleof pharmaceutically acceptable salt of hydroxychloroquine ishydroxychloroquine sulfate.

Hydroxychloroquine (HCQ) is currently used in treatments of malaria,lupus erythematosus, rheumatoid arthritis, post-Lyme disease arthritis,and Sjogren's syndrome, typically at a daily dose of 200 mg or 400 mghydroxychloroquine sulfate.

In exemplary embodiments, the hydroxychloroquine is in a form ofhydroxychloroquine sulfate (a pharmaceutically acceptable salt ofhydroxychloroquine).

Herein, weight amounts of hydroxychloroquine or a pharmaceuticallyacceptable salt thereof refer to an amount of hydroxychloroquine sulfatewhich includes the intended amount of hydroxychloroquine per se, inaccordance with the widespread use of the sulfate salt in the art. Theskilled person will be readily capable of determining an amount offree-base HCQ, or a salt of HCQ other than HCQ sulfate, which willcomprise the same amount of HCQ per se as in the recited amount of HCQsulfate.

Due to its lower toxicity compared to chloroquine, high peak levels ofHCQ are considered to be tolerable.

As demonstrated herein, it has been surprisingly found that HCQ inhibitsHCV-induced processes associated with autophagy, and is more effectivethan chloroquine at inhibiting such processes, and is also effective inmodels of antiviral resistant HCV genotypes.

Without being bound by any particular theory, it is believed thathydroxychloroquine is particularly effective at inhibiting HCVreplication by inhibiting HCV-induced autophagy which facilitates HCVreplication, and that HCQ can thereby exhibit a strong antiviral effectin an HCV-infected subject.

As used herein, the term “therapeutically effective amount” describes anamount of a compound described herein (alone or in a combination ofcompounds described herein) which upon being administered will relieveto some extent one or more of the symptoms of the condition beingtreated.

In the context of some embodiments of the present invention, a“therapeutically effective amount” describes an amount which eradicatesor reduces HCV replication. Such an amount can also be defined herein asan amount that prevents an acute HCV infection from turning into achronic HCV infection.

As indicated herein, embodiments of this aspect of the present inventionrelate to an amount which inhibits HCV-induced autophagy. Such an amountcan be determined, for example, by measuring autophagy in anHCV-infected subject (e.g., as described herein) before and afteradministration of an amount of HCQ, so as to determine whether adecrease (e.g., a decrease described herein) in HCV-induced autophagy isdetected.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof ranges between 100 mg to 2000 mg per day, including anyinteger within this range. In some embodiments, the therapeuticallyeffective amount of HCQ or a salt thereof ranges from about 400 mg toabout 2000 mg per day. In some embodiments, the therapeuticallyeffective amount of HCQ or a salt thereof ranges from about 500 mg toabout 1000 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 100 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 200 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 300 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 400 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 500 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 600 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 700 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 800 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 900 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 1000 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 1100 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 1200 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 1300 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 1400 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 1500 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt to thereof is 1600 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 1700 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 1900 mg per day.

In some embodiments, the therapeutically effective amount of HCQ or asalt thereof is 2000 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required to exert currently knowntherapeutic effects, including the amount known to date to betherapeutically effective in the treatment of malaria, lupuserythematosus, rheumatoid arthritis, post-Lyme disease arthritis andSjogren's syndrome, and including an amount considered effective fortreating HCV infections.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required to exert currently knowntherapeutic effects by at least 10%, and can be, for example, higher byfrom 10% to about 50% or from about 10% to about 100% or even 200%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by at 20%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required to exert currently knowntherapeutic effects by 30%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 40%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 50%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 60%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 70%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 80%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 90%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 100%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 110%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 120%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 150%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 200%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 300%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 400%.

In some embodiments, the amount of HCQ sufficient to inhibit autophagyis higher than the amount of HCQ required for exerting currently knowntherapeutic effects by 500%.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy is at least 400 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy is at least 500 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy is at least 600 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy is at least 700 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy is at least 800 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy is at least 900 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy is at least 1000 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy ranges from 500 mg to 1500 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy ranges from 600 mg to 1200 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy ranges from 800 mg to 1200 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy ranges from 600 mg to 1000 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy ranges from 800 mg to 1000 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy ranges from 900 mg to 1100 mg per day.

In some embodiments, the amount of HCQ sufficient to inhibitHCV-autophagy ranges from 850 mg to 950 mg per day.

Any integer between the above-indicated ranges is contemplated.

Herein throughout, whenever an amount of HCQ is indicated, itencompasses the same amount of an HCQ pharmaceutically acceptable saltas described herein, or an equimolar amount of an HCQ pharmaceuticallyacceptable salt.

Herein throughout, whenever an amount of HCQ or of a salt thereof isindicated as an amount per day, it can be administered once, twice,thrice and even four-times a day.

In some embodiments, the method is effected by administering atherapeutically effective amount of HCQ or a salt thereof once a day.

In some embodiments, the method is effected by administering atherapeutically effective amount of HCQ or a salt thereof once a day.

When administered more than once a day (e.g., twice or thrice a day),the above-indicated amounts are divided to the respective administrationtimes.

For example, in embodiments in which the method is effected byadministering an amount of HCQ which is 900 mg per day, and comprises 2daily administrations, 450 mg of HCQ are used in each administration.Alternatively, one administration is of 400 mg and another is of 500 mg.If such a daily dosage is to be administered 3 times a day, 300 mg ofHCQ can be used, as an example, in each administration.

In embodiments in which the method is effected by administering anamount of HCQ which is 600 mg per day, 300 mg of HCQ are used in each oftwo daily administrations. Alternatively, one administration is of 200mg and another is of 400 mg. Alternatively, 200 mg of HCQ are used ineach of three daily administrations.

In embodiments in which the method is effected by administering anamount of HCQ which is 800 mg per day, and comprises 2 dailyadministrations, 400 mg of HCQ are used in each administration.Alternatively, one administration is of 200 mg and another is of 600 mg.Alternatively, 200 mg of HCQ are used in each of four dailyadministrations.

In embodiments in which the method is effected by administering anamount of HCQ which is 1000 mg per day, and comprises 2 dailyadministrations, 500 mg of HCQ are used in each administration.Alternatively, one administration is of 400 mg and another is of 600 mg.Alternatively, the method comprises 3 daily administrations, forexample, two of 400 mg and one of 200 mg. Alternatively, the methodcomprises 4 daily administrations, for example, wherein eachadministration is of 250 mg.

The method described herein can be utilized for treating an HCV-relateddisease by administering HCQ or salt thereof, as described herein.However, either or both the therapeutic effect of HCQ in treatingHCV-related diseases and the effect of HCQ in inhibiting HCV-inducedautophagy can further be utilized in a combined therapy, where HCQ orthe salt thereof is used in combination with one or more anti-viralagents.

As demonstrated herein, such a combined therapy provides for at least anadditive therapeutic effect exhibited by both agents and in someembodiments provides for a synergistic therapeutic effect, as describedin further detail hereinafter.

In some embodiments, such a combined therapy provides for enhancing theeffect of the antiviral agent, via the inhibition of the HCV-inducedautophagy.

Without being bound by any particular theory, it is suggested that theupregulated autophagy demonstrated herein in various HCV models reducesthe therapeutic effect of the anti-viral agent, and therefore inhibitingthe HCV-induced autophagy increases the therapeutic effect of theanti-viral agent and can even be regarded, at least in some cases, assensitizing the HCV towards the anti-viral agent.

Thus, in some embodiments, the method described herein is furthereffected by co-administering to the subject (e.g., an HCV-infectedsubject) HCQ or a salt thereof in an amount as indicated hereinabove,and a therapeutically effective amount of at least one antiviral agent.

Herein, the term “antiviral agent” encompasses any active compound ormixture of active compounds which is active against viruses, inparticular HCV, and includes, but is not limited to, ribavirin andderivatives and prodrugs thereof (e.g., viramidine); interferons (e.g.,interferon-α); viral protease inhibitors (e.g., boceprevir, SCH 503034,telaprevir, ITMN B, BILN 2061, SCH 6); NS4A inhibitors (e.g., GS-9132);NS5A inhibitors; viral polymerase inhibitors, including nucleoside andnon-nucleoside polymerase inhibitors (e.g., NM-107 and its prodrugvalopicitabine (NM-283), R1626/R1479, HCV-796, BILB 1941, R7128/PSI6130,GSK625433, A-848837, BCX-4678, GL59728, GL60667, NV-008, HCV-086, R803,JTK 003, XTL-2125); cyclophilin B inhibitors (e.g., alisporivir(DEBIO-025), NIM811); helicase inhibitors (e.g., QU665); glycosylationinhibitors (e.g., celgosivir (MX-3253)); an antiphospholipid antibody(e.g., bavituximab); and any combination thereof.

In some embodiments, the at least one anti-viral agent is selected fromthe group consisting of a ribavirin, a viral protease inhibitor, a viralpolymerase inhibitor, an NS4A inhibitor, and a NS5A inhibitor.

The term “anti-viral agent” as used herein encompasses prodrugs,pharmaceutically acceptable salts, hydrates, solvates andpharmaceutically active derivatives of any of the exemplary agentsdescribed herein.

Examples of antiviral agents suitable for use according to embodimentsof the invention include:

PEGylated interferon alfa-2a (e.g., PEGASYS®); Interferon alfacon-1(e.g., INFERGEN®); Natural interferon (e.g., OMNIFERON®); ALBUFERON®;Interferon beta-1a (e.g., REBIF®); Omega interferon (available fromBioMedicine); Oral interferon alpha (available from AmarilloBiosciences); Interferon gamma-1b (available from InterMune); IP-501(available from Interneuron); Merimedodib VX-497 (Vertex); Amantadine;IDN-6556 (Idun Pharma.); XTL-002 (XTL); HCV/MF59 (Chiron); Civacir(NABI); Viramidine (ICN); thymosin alfa-1 (e.g., ZADAXIN) (Sci Clone);histamine dihydrochloride (CEPLENE) (Maxim); VX 950/LY 570310(Vertex/Eli Lilly); ISIS 14803 (Isis); IDN-6556 (Idun Pharma.); JTK 003(AKROS Pharma); tarvacin (Peregrine); HCV-796 (ViroPharma); CH-6(Schering); ANA971 (ANADYS); ANA245 (ANADYS); CPG 10101 (ACTILON)(Coley); rituximab; valopicitabine; HepX-C antibody (XTL); IC41(Intercell); Medusa Interferon (Flamel Technologies); E-1(Innogenetics); Multiferon (Viragen); and BILN 2061(Boehringer-Mannheim).

In some embodiments, the antiviral agent is interferon-α, as definedherein, including a prodrug thereof.

In some embodiments, the antiviral agent is ribavirin or a prodrugthereof (e.g., viramidine).

In some embodiments, the antiviral agent is boceprevir or a prodrugthereof.

In some embodiments, the antiviral agent is telaprevir.

In some embodiments, the antiviral agent is alisporivir.

In some embodiments, the antiviral agent is NM-107 or a prodrug thereof.

The term “prodrug” refers to an agent, which is converted into theactive compound (the active parent drug) in vivo. Prodrugs are typicallyuseful for facilitating the administration of the parent drug. They may,for instance, be bioavailable by oral to administration whereas theparent drug is not. The prodrug may also have improved solubility ascompared with the parent drug in pharmaceutical compositions. Prodrugsare also often used to achieve a sustained release of the activecompound in vivo.

The term “solvate” refers to a complex of variable stoichiometry (e.g.,di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by asolute (the compound of present invention) and a solvent, whereby thesolvent does not interfere with the biological activity of the solute.Suitable solvents include, for example, ethanol, acetic acid and thelike.

The term “hydrate” refers to a solvate, as defined hereinabove, wherethe solvent is water.

As used herein, the term “co-administering” describes administering tothe subject two agents during the treatment. This term encompassesadministering the anti-viral agent prior to, concomitant with orsubsequent to administering the HCQ or the salt thereof. This term alsoencompasses administering the two agents via the same route ofadministration or via different routes of administration. This termfurther encompasses administering the two agents within a singlepharmaceutical composition or in two separate pharmaceuticalcompositions, each comprising a single agent, as is further detailedhereinbelow.

In some embodiments, co-administering is effected such that the efficacywindow of the HCQ and the efficacy window of the antiviral agentsubstantially overlap.

As is well known in the art, an efficacy window of an agent depends onvarious factors such as systemic absorbance rate, the time required toreach a plasma peak concentration and/or clearance rate.

It is often desirable to treat subjects suffering from an HCV-relateddisease with two or more compounds which exhibit an antiviral effect, soas to simultaneously act on the virus via two or more antiviralmechanisms, when one compound sensitizes HCV to the second compound,and/or when the compounds act in synergy.

Simultaneous action of two or more agents can be achieved if the agentsexhibit their effect within the same time frame.

As used herein, the phrase “efficacy window” describes a time frameduring which an active agent exhibits a desired pharmacological effect,such as an antiviral effect, upon administration. In other words, thisphrase describes that time period at which the plasma concentration ofan active agent is equal to or higher than a minimal pharmacologicallyeffective concentration thereof.

The phrase “substantially overlap” with respect to the efficacy windowsof the active agents means that during a certain time period uponadministration of two agents described herein (e.g., HCQ and anantiviral agent), both agents exhibit a desired pharmacological effectto some extent, namely, a plasma concentration of each agent is equal toor is higher than a minimum pharmacologically effective concentration ofthe agent. The efficacy windows of the active agents can overlap for,for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes,30 minutes, 1 hour, 2 hours, 3 hours and even for longer time periods.The efficacy windows of the active agents can overlap such that duringthe overlapping period, both agents exhibit a maximal efficacy, suchthat one agent exhibits a maximal efficacy while the other agentexhibits a partial efficacy or such that both agents exhibit a partialefficacy.

In some embodiments, the anti-viral agent is an interferon.

In some embodiments, the antiviral agent comprises an interferon and atleast one other antiviral agent described herein (e.g., an antiviralagent that does not inhibit HCV-induced autophagy). Optionally HCQ isco-administered with an interferon and ribavirin.

As used herein, the term “interferon” refers to a member of a family ofhighly homologous species-specific proteins that inhibit viralreplication and cellular proliferation and modulate immune response.Herein, the term “interferon” encompasses derivatives of the naturallyoccurring proteins, including, without limitation, mutant forms of anaturally occurring interferon, and derivatives (e.g., conjugates) of anaturally occurring interferon, such as PEGylated interferon(polyethylene glycol modified conjugates of interferon) and interferonattached to another protein (e.g., as a fusion protein).

Human interferons are grouped into three classes based on their cellularorigin and antigenicity: interferon-α (leukocytes), interferon-β(fibroblasts) and interferon-γ (B cells), each of which is encompassedherein by the term “interferon”. Recombinant to forms of each group havebeen developed and are commercially available. Subtypes in each groupare based on antigenic/structural characteristics.

In exemplary embodiments, the interferon is an interferon-α.

As used herein, the term “interferon-α” refers to a class ofinterferons. At least 24 versions of interferon-α (grouped into subtypesA through H), having distinct amino acid sequences, have been identifiedby isolating and sequencing DNA encoding these peptides. Both naturallyoccurring and recombinant interferon-α, including consensus interferon,may be used in the practice of the invention. Suitable interferon-α forthe present of embodiments of the invention described herein includes,but is not limited to, recombinant interferon alfa-2b such as INTRON®-Ainterferon and VIRAFERON®; recombinant interferon alfa-2a such asROFERON® interferon; recombinant interferon alfa-2c such as BEROFOR®alfa 2 interferon; interferon alfa-n1, a purified blend of natural alfainterferons such as SUMIFERON® or WELLFERON® interferon alfa-n1 (INS);or a consensus alfa interferon such as those described in U.S. Pat. Nos.4,897,471 and 4,695,623; or interferon alfa-n3, a mixture of naturalalfa interferons such as ALFERON®. In some embodiments, the interferon-αis selected from the group consisting of interferon alfa-2a andinterferon alfa 2b. The manufacture of interferon alfa 2b is describedin U.S. Pat. No. 4,530,901.

The term “interferon-α” is further intended to include interferonderivatives such as PEGylated analogs of interferon-α, optionallyinterferon alfa-2a and -2b. PEGylated interferon-α that may be used inembodiments of the present invention are, e.g., PEGylated interferonalfa-2a, PEGylated interferon alfa-2b, PEGylated consensus interferonand PEGylated purified interferon alfa product. PEGylated interferonalfa-2a is described, e.g., in European Patent No. EP 0593868 andcommercially-available, e.g., under the tradename PEGASYS® (Hoffmann-LaRoche). PEGylated interferon alfa-2b is described, e.g., in U.S. Pat.No. 5,908,621 and WO 98/48840 and commercially-available, e.g., underthe tradename PEGINTRON® A (Schering Plough). PEGylated consensusinterferon is described in WO 96/11953. The term “interferon-α” furtherencompasses other interferon-α conjugates that can be prepared bycoupling an interferon-α to a water-soluble polymer. A non-limiting listof such polymers includes other polyalkylene oxide homopolymers such aspolyethylene glycol (PEG), polypropylene glycols, polyoxyethylenatedpolyols, copolymers thereof and block to copolymers thereof. As analternative to polyalkylene oxide-based polymers, effectivelynon-antigenic materials such as dextran, polyvinylpyrrolidones,polyacrylamides, polyvinyl alcohols, carbohydrate-based polymers and thelike can be used. Such interferon-α-polymer conjugates are described inU.S. Pat. No. 4,766,106, U.S. Pat. No. 4,917,888, European PatentApplication No. 0236987, European Patent Application Nos. 0510356,0593868 and 0809996 (PEGylated interferon alfa-2a) and InternationalPublication No. WO 95/13090.

The term “interferon-α” further includes fusion proteins of aninterferon-α, for example fusion proteins of interferon-α-2a,interferon-α-2b, consensus interferon or purified interferon-α product,each of which is fused with another protein. Certain preferred fusionproteins comprise an interferon (e.g., interferon-α-2b) and an albuminas described in U.S. Pat. No. 6,972,322 and international patentapplication publications WO2005/003296 and WO2005/077042. An optionalinterferon conjugated to a human albumin is ALBUFERON® which is alonger-acting form of interferon-α created using albumin fusiontechnology. ALBUFERON® results from the genetic fusion of human albuminand interferon-α. Also included are consensus interferons, such asINFERGEN®.

In exemplary embodiments, the interferon is PEGylated interferon α-2a.

As demonstrated in the Examples section that follows, it has been shownthe HCQ and an interferon-α can act in synergy.

By “synergy” it is meant that the effect of the compounds whenadministered in combination is greater than an additive effect of thecompounds when administered alone as a single agent.

As exemplified in the Examples below, synergy can be determinedaccording to methods described, for example, by Prichard & Shipman[Antiviral Res 1990, 14:181-205], wherein the theoretical additiveeffect is calculated from dose-response curves of individual compoundsby the equation Z=X+Y(1−X), where X and Y represent the inhibitionproduced by the individual compounds and Z represents the effectproduced by the combination of compounds. An effect of a combination ofcompounds which is higher than Z (optionally 20% higher, and optionally30% higher) indicates synergism.

As further exemplified in the Examples below, synergy can be determinedaccording to methods described by Chou & Talalay [Trends Pharmacol Sci.1983, 4:450-454; Adv Enzyme Regul 1984, 22:27] and/or using anisobologram, e.g., as described by Tallarida [J Pharmacol Exp Therap2001, 298:865-872].

In some embodiments, a synergistic effect is determined according to theabovementioned method of Prichard & Shipman. In exemplary someembodiments, a synergistic effect is determined according to each of theabovementioned methods.

Accordingly, in some embodiments, the method as described herein iseffected by co-administering to the subject, as defined herein, anamount of HCQ as defined herein and a therapeutically effective amountof an interferon as defined herein.

In some embodiments, the therapeutically effective amount of theinterferon (e.g., PEGylated interferon) ranges from 50 to 250 μg(optionally per week).

In some embodiments, the therapeutically effective amount of interferonis in a range of from 3,000-1,000,000 units, optionally, from10,000-1,000,000 units, per administration (optionally per week).

The frequency of interferon administration may depend, at least in part,on the half-life of the interferon in a body. PEGylated interferonstypically have a longer half-life, and are therefore administered lessfrequently than other interferons.

In some embodiments, an interferon (e.g., a non-PEGylated interferon) isadministered from 2-4 times per week, optionally 3 times per week.

In some embodiments, a PEGylated interferon is administered once perweek.

In some embodiments, the method is effected by co-administering to thesubject an amount of HCQ as described herein and a therapeuticallyeffective amount of ribavirin (or a prodrug thereof).

In some embodiments, the method is effected by co-administering to thesubject an amount of HCQ as described herein and a therapeuticallyeffective amount of boceprevir.

In some embodiments, the method is effected by co-administering to thesubject an amount of HCQ as described herein and a therapeuticallyeffective amount of telaprevir.

A current standard treatment of HCV-related diseases comprisesco-administration of ribavirin with an interferon-α (e.g., a PEGylatedinterferon-α).

Hence, in some embodiments, the method is effected by co-administeringto the to subject an amount of HCQ as described herein and atherapeutically effective amount of ribavirin and a therapeuticallyeffective amount of an interferon-α (e.g., a PEGylated interferon-α).

In some embodiments, the method is effected by co-administering to thesubject an amount of HCQ as described herein and a therapeuticallyeffective amount of ribavirin and a therapeutically effective amount ofboceprevir.

In some embodiments, the method is effected by co-administering to thesubject an amount of HCQ as described herein and a therapeuticallyeffective amount of ribavirin and a therapeutically effective amount oftelaprevir.

In some embodiments, the method is effected by co-administering to thesubject an amount of HCQ as described herein and a therapeuticallyeffective amount of ribavirin, a therapeutically effective amount of aninterferon-α, and a therapeutically effective amount of boceprevir.

In some embodiments, the method is effected by co-administering to thesubject an amount of HCQ as described herein and a therapeuticallyeffective amount of ribavirin, a therapeutically effective amount of aninterferon-α, and a therapeutically effective amount of telaprevir.

In some embodiments, the method is effected by co-administering to thesubject an amount of HCQ as described herein and a therapeuticallyeffective amount of ribavirin, a therapeutically effective amount of aninterferon-α, and a therapeutically effective amount of an NS5Binhibitor.

In some embodiments, the method is effected by co-administering to thesubject an amount of HCQ as described herein and a therapeuticallyeffective amount of ribavirin, a therapeutically effective amount of aninterferon-α, and a therapeutically effective amount of a cyclophilininhibitor (e.g., a cyclophilin inhibitor described herein).

In some embodiments, the method is effected by co-administering to thesubject an amount of HCQ as described herein and a therapeuticallyeffective amount of ribavirin, a therapeutically effective amount of aninterferon-α, and a therapeutically effective amount of an NS5Ainhibitor.

As described herein, it is often desirable to treat subjects sufferingfrom an HCV-related disease with two or more compounds which exhibitcomplementary effects.

Hence, in some embodiments, HCQ, which inhibits HCV-induced autophagy,is co-administered with an antiviral agent which acts against HCV by adifferent mechanism, e.g., wherein the antiviral agent does not inhibitHCV-induced autophagy.

Examples of suitable anti-viral agent which do not inhibit HCV-inducedautophagy include, without limitation, polymerase inhibitors (e.g.,NM-107, valopicitabine), ribavirin, viramidine, boceprevir, telaprevir,and alisporivir. Ribavirin is an exemplary antiviral agent. In someembodiments, a prodrug of ribavirin (e.g., viramidine) is used.

Without being bound by any particular theory, it is believed that theinhibition of autophagy by HCQ disrupts the life cycle of HCV to anextent which renders the HCV considerably more susceptible to a secondantiviral effect (e.g., an antiviral effect of a antiviral agentco-administered with HCQ), such that the HCQ may sensitize HCV to theantiviral agent, for example, HCV which is partially or fully resistantto the antiviral agent.

Further according to an aspect of some embodiments of the presentinvention there is provided hydroxychloroquine which is identified foruse in the treatment of a hepatitis C virus (HCV) related disease in anamount sufficient to inhibit HCV-induced autophagy, as defined herein.

According to embodiments of this aspect of the present invention the HCQis for use in the treatment of an HCV-related disease, as describedherein, wherein the treatment is effected by administering to a subjectin need thereof, as described herein, an amount of HCQ sufficient toinhibit HCV-induced autophagy, as defined herein.

Further according to an aspect of some embodiments of the presentinvention there is provided a use of a hydroxychloroquine in themanufacture of a medicament for use in the treatment of a hepatitis Cvirus (HCV) related disease. In some embodiments, the medicament isidentified for use in treating HCV related disease by administering to asubject in need thereof hydroxychloroquine in an amount sufficient toinhibit HCV-induced autophagy, as defined herein.

In some embodiments of this aspect of the present invention, themedicament comprises a therapeutically effective amount of HCQ or a saltthereof, or, the medicament comprises instructions to administer to thesubject an amount of HCQ, as indicated herein.

The amount of HCQ sufficient to inhibit HCV-induced autophagy isoptionally any amount or range of amounts described elsewhere herein(e.g., from 400 to 2000 mg per day).

HCV-induced autophagy is optionally characterized as described elsewhereherein, and optionally by an increase in a level of protein selectedfrom the group consisting of ULK1, AMBRA1, ATG2A, GABARAPL1, FOX03,SQSTM1, PIK3C3 and MAP1LC3B (e.g., as described herein).

In some embodiments, the disease is caused by an antiviral-resistant HCV(e.g., as described elsewhere herein), and optionally by genotype 1 HCVand/or genotype 4 HCV.

The abovementioned treatment may be effected in accordance with any ofthe methods described herein, and optionally further comprisesco-administering a therapeutically effective amount of an antiviralagent (e.g., any individual antiviral agent or combination of antiviralagents described herein for co-administration with HCQ).

As discussed herein, it is believed that the inhibition of autophagy byHCQ disrupts the life cycle of HCV to an extent which renders the HCVconsiderably more susceptible to a different antiviral effect (e.g., anantiviral effect which does not involve inhibition of autophagy), suchthat the HCQ may sensitize HCV to a antiviral agent, particularly anantiviral agent which does not inhibit HCV-induced autophagy.

Hence, according to an aspect of some embodiments of the presentinvention there is provided a method of treating an hepatitis C virus(HCV) related disease, such as an HCV infection, as defined herein, in asubject in need thereof, which is effected by co-administering to thesubject a therapeutically effective amount of hydroxychloroquine or apharmaceutically acceptable salt thereof and a therapeutically effectiveamount of an antiviral agent, wherein the anti-viral agent does notinhibit HCV-induced autophagy, thereby treating the HCV infection.

In some embodiments, the therapeutically effective amount ofhydroxychloroquine is sufficient to inhibit HCV-induced autophagy in thesubject. Suitable amounts according to some embodiments of the inventionare described elsewhere herein.

Examples of suitable anti-viral agent which do not inhibit HCV-inducedautophagy include, without limitation, ribavirin, viramidine,boceprevir, telaprevir, and alisporivir. Ribavirin is an exemplaryantiviral agent. In some embodiments, a prodrug of ribavirin (e.g.,viramidine) is used.

In some embodiments, the method further comprises co-administering tothe subject a therapeutically effective amount of an additionalantiviral agent, such that at least 3 compounds are co-administered: HCQand at least two antiviral agents, namely, the above-describedanti-viral agent which does not inhibit HCV-induced autophagy, and theaforementioned additional antiviral agent.

The additional antiviral agent may be, for example, any antiviral agentdescribed herein (with the proviso that it is not identical to theaforementioned anti-viral agent which does not inhibit HCV-inducedautophagy), and it may or may not be capable of inhibiting HCV-inducedautophagy. Thus, for example, the additional antiviral agent may beanyone of an interferon, a viral protease inhibitor, an NS4A inhibitor,an NS5A inhibitor, a viral polymerase inhibitor, a cyclophilininhibitor, a helicase inhibitor, a glycosylation inhibitor, and anantiphospholipid antibody (e.g., as described elsewhere herein).

Co-administration of particular combinations of HCQ and two antiviralagents, wherein at least one antiviral agent does not inhibitHCV-induced autophagy, are described elsewhere herein, and are suitablefor use according to some embodiments of this aspect of the invention.

In some embodiments, the additional antiviral agent is an interferon(e.g., an interferon described herein). Optionally, the interferon is aninterferon-α, and optionally a PEGylated interferon-α (e.g., aninterferon-α described herein).

In some embodiments, the aforementioned interferon is co-administeredwith ribavirin, such that the method comprises co-administering HCQ, theinterferon and ribavirin. The therapeutically effective amount ofribavirin is optionally in a range of from 50 to 1200 mg per day(optionally from 50 to 150 mg per day; and optionally from 400 to 1200mg per day).

Further according to an aspect of some embodiments of the presentinvention to there is provided hydroxychloroquine which is identifiedfor use in the treatment of a hepatitis C virus (HCV) related disease,as defined herein, in combination with an antiviral agent that does notinhibit HCV-induced autophagy, as defined herein.

According to embodiments of this aspect of the present invention the HCQis for use in the treatment of an HCV-related disease, as describedherein, wherein the treatment is effected by administering to a subjectin need thereof, as described herein, HCQ in combination with anantiviral agent that does not inhibit HCV-induced autophagy, as definedherein.

The treatment can be effected by co-administering the two agentsseparately or co-formulated in a single pharmaceutical composition, asis further detailed hereinafter.

Further according to an aspect of some embodiments of the presentinvention there is provided a use of a hydroxychloroquine in themanufacture of a medicament for use in the treatment of a hepatitis Cvirus (HCV) related disease. In some embodiments, the medicament isidentified for use in treating HCV related disease by administering to asubject in need thereof hydroxychloroquine in combination with anantiviral agent that does not inhibit HCV-induced autophagy, as definedherein.

In some embodiments of this aspect of the present invention, themedicament comprises a therapeutically effective amount of HCQ or a saltthereof, or, the medicament comprises instructions to administer to thesubject an amount of HCQ, as indicated herein.

In some embodiments, the medicament further comprises a therapeuticallyeffective amount of an antiviral agent that does not inhibit HCV-inducedautophagy, or, the medicament comprises instructions to co-administer tothe subject an amount of an antiviral agent that does not inhibitHCV-induced autophagy, as indicated herein.

In some embodiments, the medicament comprises HCQ and an anti-viralagent as described herein which are co-formulated in a singlepharmaceutical composition, as is further detailed hereinbelow.

The therapeutically effective amount of HCQ according to someembodiments of this aspect of the invention is an amount of HCQ or rangeof amounts of HCQ described elsewhere herein (e.g., from 400 to 2000 mgper day).

In some embodiments of any of the aspects of the invention describedherein which relate to co-administration of HCQ with one or moreantiviral agents, the disease to be treated is caused by anantiviral-resistant HCV, and optionally by an HCV genotype that isresistant to an antiviral agent co-administered with the HCQ (e.g., anantiviral agent which does not inhibit HCV-induced autophagy, as definedherein). In some embodiments, the disease is caused by genotype 1 HCVand/or genotype 4 HCV.

The abovementioned treatment may be effected in accordance with any ofthe methods described herein, and optionally further comprisesco-administering a therapeutically effective amount of an antiviralagent (e.g., any individual antiviral agent or combination of antiviralagents described herein for co-administration with HCQ).

As discussed hereinabove, HCQ exhibits effects (e.g., inhibition ofHCV-induced autophagy) which can enhance the efficacy of an antiviralagent, including, for example, sensitizing an HCV towards an anti-viralagent to which the HCV is resistant in the absence of HCQ.

Hence, according to an aspect of some embodiments of the presentinvention there is provided a method of treating an hepatitis C virus(HCV) related disease in a subject in need thereof, wherein the diseaseis caused by an HCV genotype resistant to an antiviral agent. Themethod, according to this aspect of embodiments of the invention, iseffected by co-administering to the subject a therapeutically effectiveamount of hydroxychloroquine or a pharmaceutically acceptable saltthereof and a therapeutically effective amount of the antiviral agent,to which the HCV genotype is resistant.

Herein, the phrase “HCV genotype resistant to an antiviral agent” and“hepatitis C virus (HCV) genotype resistant to an antiviral agent” referto an HCV genotype in an infected subject which was determined to beresistant to a given antiviral agent by: a) prior treatment of thesubject with the antiviral agent, wherein the subject was non-responsiveto the treatment; and/or b) identification of the genotype of the HCV,wherein the genotype is identified as one wherein most (>50%) subjectsinfected with such an HCV genotype are non-responsive to treatment withthe antiviral agent. The phrase “resistant to an antiviral agent” doesnot include resistance which can be overcome by simply raising a dosageof the antiviral agent to a higher dosage which is still tolerated bythe subject.

In some embodiments, the disease is caused by genotype 1 HCV and/orgenotype 4 HCV.

In some embodiments, subjects treatable by the method as describedherein are subjects afflicted by HCV, which were identified as afflictedby a resistant genotype of HCV, and/or subjects afflicted by HCV whichreceived one or more cycles of antiviral therapy but were foundnon-responsive to this therapy in terms of no or incomplete eradicationof the viral infection and/or in terms of insufficient relief ofsymptoms associated with the viral infection.

The therapeutically effective amount of the antiviral agent which isco-administered according to this aspect of embodiments of the inventionmay optionally be an amount which is effective for treating anon-resistant HCV genotype. Alternatively or additionally, thetherapeutically effective amount is an amount which exerts a desiredtherapeutic effect when co-administered with HCQ. Optionally, thetherapeutically effective amount is selected to be tolerated by thesubject.

It is to be appreciated that embodiments according to this aspect of theinvention involve sensitization of an HCV genotype to an antiviralagent, such that the antiviral agent exerts a clinically significantantiviral effect on the HCV when co-administered with HCQ as describedherein, whereas such an antiviral effect would not be exerted whenadministered without HCQ.

The therapeutically effective amount of HCQ can be regarded as asensitizing effective amount.

Herein, “sensitizing effective amount” refers to an amount of HCQ whichcauses an HCV genotype resistant to an antiviral agent (as definedherein) to be susceptible (i.e., no longer resistant) to aco-administered amount of the antiviral agent.

In some embodiments, the therapeutically effective amount ofhydroxychloroquine is sufficient to inhibit HCV-induced autophagy (asdefined herein) in the subject, as described elsewhere herein.

Suitable therapeutically effective amounts according to some embodimentsof the invention are described elsewhere herein.

In some embodiments, the antiviral agent does not inhibit HCV-inducedautophagy. Suitable examples of such antiviral agents are describedelsewhere herein.

In some embodiments, the antiviral agent is ribavirin. In someembodiments, the antiviral agent is a prodrug of ribavirin (e.g.,viramidine).

In some embodiments, the method further comprises co-administering tothe subject a therapeutically effective amount of an additionalantiviral agent, such that at least 3 compounds are co-administered: HCQand at least two antiviral agents, namely, the above-describedanti-viral agent which does not inhibit HCV-induced autophagy, and theaforementioned additional antiviral agent, as described in more detailelsewhere herein.

Further according to an aspect of some embodiments of the presentinvention there is provided hydroxychloroquine which is identified foruse in combination with an antiviral agent in the treatment of ahepatitis C virus (HCV) related disease caused by an HCV genotyperesistant to the antiviral agent (as defined herein).

According to embodiments of this aspect of the present invention the HCQis for use in the treatment of an HCV-related disease, as describedherein, wherein the treatment is effected by administering to a subjectin need thereof, as described herein, HCQ in combination with anantiviral agent, wherein the disease is caused by an HCV genotyperesistant to the antiviral agent, as defined herein.

Further according to an aspect of some embodiments of the presentinvention there is provided a use of a hydroxychloroquine in themanufacture of a medicament for use in the treatment of a hepatitis Cvirus (HCV) related disease caused by an HCV genotype resistant to anantiviral agent, as defined herein. In some embodiments, the medicamentis identified for use in treating an HCV related disease byadministering to a subject in need thereof hydroxychloroquine incombination with the aforementioned antiviral agent.

The antiviral agent may optionally be any antiviral agent describedherein, including combinations of antiviral agents described herein. Insome embodiments, the antiviral agent is an interferon-α and/orribavirin, such that the HCV genotype is resistant to interferon-αand/or ribavirin.

In some embodiments of this aspect of the present invention, themedicament comprises a therapeutically effective amount (e.g., asensitizing effective amount, as defined herein) of HCQ or a saltthereof, or, the medicament comprises instructions to administer to thesubject a therapeutically effective amount of HCQ, as indicated herein.

In some embodiments, the abovementioned treatment comprisesadministration of HCQ in an amount sufficient to inhibit HCV-inducedautophagy, as described elsewhere herein.

The abovementioned treatment may be effected in accordance with methodsdescribed herein.

The therapeutically effective amount of HCQ according to someembodiments of this aspect of the invention is an amount of HCQ or rangeof amounts of HCQ described elsewhere herein (e.g., from 400 to 2000 mgper day).

Optionally, the antiviral agent is an antiviral agent that does notinhibit HCV-induced autophagy (e.g., ribavirin), as described elsewhereherein.

In some embodiments, the disease is caused by genotype 1 HCV and/orgenotype 4 HCV.

As exemplified in the Examples section below, HCQ exhibits a synergisticantiviral effect when in combination with another antiviral agent, andsuch synergistic effects are surprisingly not exhibited by thestructurally similar chloroquine. Such a synergistic effect enhances thetherapeutic potential of both HCQ and the antiviral agent, and therebyallows for more effective antiviral treatment for any given drug dosageand/or treatment at lower dosages which results in better tolerance andfewer adverse effects.

Hence, according to another aspect of embodiments of the presentinvention, there is provided method of treating a hepatitis C virus(HCV) related disease in a subject in need thereof, the methodcomprising co-administering to the subject a therapeutically effectiveamount of hydroxychloroquine or a pharmaceutically acceptable saltthereof in combination with a therapeutically effective amount of anantiviral agent, wherein the therapeutically effective amount ofhydroxychloroquine and the therapeutically effective amount of theantiviral agent are selected such that hydroxychloroquine and theanti-viral agent act in synergy, as defined herein.

Combinations which act in synergy are also referred to herein assynergistic combinations.

Therapeutically effective amounts which result in synergy may beselected by determining the effects of different combinations of HCQ andthe antiviral agent to (optionally including the effects of HCQ aloneand/or the antiviral agent alone), as exemplified in the Examplessection.

In some embodiments, synergy is effected using an amount of HCQ which isat least 400 mg per day (e.g., in a range of from 400 to 2000 mg perday), preferably at least 500 mg per day (e.g., from 500 to 1000 mg perday), optionally at least 600 mg per day, and optionally at least 800 mgper day (e.g., 800-1000 mg/day, 850-950 mg/day, 900 mg/day). Suitableranges of at least 600 mg per day and at least 800 mg per day aredescribed elsewhere herein.

The antiviral agent exhibiting synergy in combination with HCQ isoptionally any antiviral agent or combination of antiviral agentsdescribed herein.

In some embodiments, the antiviral agent exhibiting synergy incombination with HCQ is a viral protease inhibitor (e.g., a proteaseinhibitor described herein).

In some embodiments, the antiviral agent exhibiting synergy incombination with HCQ is a viral polymerase inhibitor (e.g., a polymeraseinhibitor described herein).

In some embodiments, the antiviral agent exhibiting synergy incombination with HCQ is an interferon. In exemplary embodiments, theinterferon is an interferon-α (e.g., PEGylated interferon-α).

In some embodiments, the therapeutically effective amount of aninterferon is in a range of from 50 to 250 μg per administration(optionally per week), as described elsewhere herein.

According to some embodiments, the method comprises co-administeringfrom 400 to 2000 mg per day of HCQ (or a pharmaceutically acceptablesalt thereof) in combination with from 50 to 250 μg of an interferon(e.g., as described herein).

In some embodiments, the therapeutically effective amount ofhydroxychloroquine, in addition to exhibiting synergy, is sufficient toinhibit HCV-induced autophagy as described herein.

Optionally, the method further comprises co-administration of atherapeutically effective amount of an additional antiviral agent (e.g.,an antiviral agent described herein), which may or may not exhibitsynergy, such that at least 3 compounds are co-administered: HCQ, and atleast two antiviral agents, at least one of which exhibits synergy incombination with HCQ, as described herein. Optionally, the additionalantiviral agent is ribavirin (or a prodrug thereof).

Further according to an aspect of some embodiments of the presentinvention there is provided hydroxychloroquine which is identified foruse in combination with an antiviral agent in the treatment of ahepatitis C virus (HCV) related disease, wherein the HCQ and theantiviral agent act in synergy (as defined herein) in treating thedisease (e.g., when co-administered as described herein).

According to embodiments of this aspect of the present invention the HCQis for use in the treatment of an HCV-related disease, as describedherein, wherein the treatment is effected by administering to a subjectin need thereof, as described herein, HCQ in combination with anantiviral agent, wherein the HCQ and the antiviral agent act in synergyin treating the disease, as described herein.

According to these embodiments, the treatment comprises a synergisticcombination of HCQ and the anti-viral agent, as defined herein.

Further according to an aspect of some embodiments of the presentinvention there is provided a use of a hydroxychloroquine in themanufacture of a medicament for use in the treatment of a hepatitis Cvirus (HCV) related disease. In some embodiments, the medicament isidentified for use in treating an HCV related disease by administeringto a subject in need thereof hydroxychloroquine in combination with anantiviral agent that acts in synergy with HCQ (e.g., as describedherein).

According to these embodiments, the treatment comprises a synergisticcombination of HCQ and the anti-viral agent, as defined herein.

Suitable antiviral agents and dosages (of HCQ and the antiviral agent)for effecting synergy are described herein.

A therapeutically effective amount of an additional antiviral agent mayoptionally be co-administered with the HCQ and with the antiviral agentwhich acts in synergy with HCQ, as described elsewhere herein.

The methods and treatments described herein according to various aspectsof the invention may comprise a step wherein one single pharmaceuticalcomposition comprising hydroxychloroquine, or a pharmaceuticallyacceptable salt thereof, an antiviral agent, and optionally at least onepharmaceutically acceptable carrier, diluent, excipients and/or additiveis administered. Alternatively, the methods of the invention maycomprise a step wherein distinct compositions comprising at least one ofthe active ingredients cited above together with one or more acceptablecarriers thereof are administered substantially simultaneously orsequentially.

The combination of the invention may be preferably administered orally.The active combined drug compounds employed in the instant therapy canbe administered in various oral forms including, but not limited to,tablets, capsules, pills, powders, granules, elixirs, tinctures,suspensions, syrups, and emulsions. It is contemplated that the activedrug compounds can be delivered by any pharmaceutically acceptable routeand in any pharmaceutically acceptable dosage form. These include, butare not limited to the use of oral conventional rapid-release, timecontrolled-release, and delayed-release pharmaceutical dosage forms. Theactive drug components can be administered in a mixture with suitablepharmaceutical diluents, excipients or carriers (collectively referredto herein as “carrier” materials suitably selected to with respect tothe intended form of administration. As indicated, it is contemplatedthat oral administration can be effectively employed. Thus, tablets,capsules, syrups, and the like as well as other modalities consistentwith conventional pharmaceutical practices can be employed.

According to another embodiment, the active ingredients used by theinvention or composition comprising a combination thereof, may beadministered via any mode of administration. For example, oral,intravenous, intramuscular, subcutaneous, intraperitoneal, parenteral,transdermal, intravaginal, intranasal, mucosal, sublingual, topical,rectal or subcutaneous administration, or any combination thereof.

In instances in which oral administration is in the form of a tablet orcapsule, the active drug components can be combined with a non-toxicpharmaceutically acceptable inert carrier such as lactose, starch,sucrose, glucose, modified sugars, modified starches, methylcelluloseand its derivatives, dicalcium phosphate, calcium sulfate, mannitol,sorbitol, and other reducing and non-reducing sugars, magnesiumstearate, stearic acid, sodium stearyl fumarate, glyceryl behenate,calcium stearate and the like. For oral administration in liquid form,the active drug components can be combined with non-toxicpharmaceutically acceptable inert carriers such as ethanol, glycerol,water and the like. When desired or required, suitable binders,lubricants, disintegrating agents and coloring and flavoring agents canalso be incorporated into the mixture. Stabilizing agents such asantioxidants, propyl gallate, sodium ascorbate, citric acid, calciummetabisulphite, hydroquinone, and 7-hydroxycoumarin can also be added tostabilize the dosage forms. Other suitable compounds can includegelatin, sweeteners, natural and synthetic gums such as acacia,tragacanth, or alginates, carboxymethylcellulose, polyethylene, glycol,waxes and the like.

Alternatively, the combination of this invention may also beadministered in controlled release formulations such as a slow releaseor a fast release formulation. Such controlled release formulations ofthe combination of this invention may be prepared using methods wellknown to those skilled in the art. The method of administration will bedetermined by the attendant physician or other person skilled in the artafter an evaluation of the subject's conditions and requirements.

The combined compounds of the present invention are generallyadministered in the form of a pharmaceutical composition comprising bothcompounds of this invention together with a pharmaceutically acceptablecarrier or diluent. Thus, the compounds used by this invention can beadministered either individually in a kit or together in anyconventional oral, parenteral or transdermal dosage form.

More particularly, since the present invention relates to the treatmentof diseases and conditions with a combination of active ingredientswhich may be administered separately, the invention also relates as afurther aspect, to combining separate pharmaceutical compositions in kitform. The kit includes two separate pharmaceutical compositions:hydroxychloroquine, or a pharmaceutically acceptable salt thereof, andan interferon alfa. The kit includes container means for containing bothseparate compositions; such as a divided bottle or a divided foil packethowever, the separate compositions may also be contained within asingle, undivided container. Typically the kit includes directions forthe administration of the separate components. The kit form isparticularly advantageous when the separate components are preferablyadministered in different dosage forms (e.g., oral and parenteral), areadministered at different dosage intervals, or when titration of theindividual components of the combination is desired by the prescribingphysician.

The kit may be for effecting any of the methods of treatment describedherein, optionally with instructions describing how to effect themethod.

It should be appreciated that both components of the kit, thehydroxychloroquine in the first dosage form and the antiviral agent inthe second dosage form may be administered simultaneously.

Alternatively, said first compound or dosage form and said secondcompound or dosage form are administered sequentially in either order.

In any of the methods and used described hereinabove, administration ofHCQ and optionally another one or more antiviral agent(s) is effectedfor a time period that optionally ranges from 1 month to life,optionally from 24 weeks to life, and optionally from 24 weeks to 1year, depending on the HCV-related disease to be treated.

Generally, administration is effected as long as virus is found in thesubject and/or until at least one of the symptoms associated with thedisease are alleviated.

In embodiments where the HCV-related is chronic, treatment is effectedfor at least 24 weeks, as described herein.

According to further aspects of some embodiments of the presentinvention there are provided pharmaceutical kits.

In some embodiments, there is provided a kit comprising HCQ andanti-viral agent, each being individually packaged within the kit,wherein the kit comprises instructions to use HCQ in an amountsufficient to inhibit HCV-induced autophagy, as defined herein.

In any of the methods, uses and kits described herein, the HCQ or a saltthereof and the anti-viral agent, if utilized, can be utilized eitherper se or can form a part of a pharmaceutical composition which furthercomprises a pharmaceutically acceptable carrier, as defined herein.

In any of the methods and uses described herein, whenever HCQ or a saltthereof is used in combination, or is co-administered, with ananti-viral agent, the HCQ and the anti-viral agent can be formulatedinto a single pharmaceutical composition.

According to further aspects of embodiments of the present inventionthere is provided a pharmaceutical composition comprising HCQ (or apharmaceutically acceptable salt thereof, such as HCQ sulfate) and ananti-viral agent (e.g., an antiviral agent described herein as beinguseful when co-administered with HCQ), and a pharmaceutically acceptablecarrier. In some embodiments, the anti-viral agent is ribavirin.

In some embodiments, the composition comprises more than one antiviralagent. Such compositions may be formulated so as to be suitable foreffecting a method of treatment described herein which comprisesco-administration of HCQ and at least two antiviral agents.

In some embodiments, the composition is formulated so as to combine theautophagy-inhibiting properties of HCQ with an antiviral agent whichacts via a different mechanism, as discussed in more detail elsewhereherein. Thus, the antiviral agent in the composition is an antiviralagent that does not inhibit HCV-induced autophagy (e.g., as describedherein). Optionally, the composition is identified for use (e.g., in oron a packaging material) for use in treating an HCV-related disease. Insome embodiments, the composition is identified for use in the treatmentof an HCV-related infection in an HCV-infected subject non-responsive toan anti-HCV therapy (e.g., as defined herein).

In some embodiments, the composition is formulated for treating anHCV-related disease caused by an HCV genotype resistant to an antiviralagent (e.g., by sensitizing HCV to the antiviral agent), as discussed inmore detail elsewhere herein. Thus, the antiviral agent in thecomposition is an antiviral agent to which an HCV genotype is resistant(e.g., as described herein). Optionally, the composition is identifiedfor use (e.g., in or on a packaging material) for use in treating anHCV-related disease caused by an HCV genotype resistant to an antiviralagent (e.g., as described herein).

In some embodiments, the composition is formulated so as to provide asynergistic effect between the HCQ and the antiviral agent, as discussedin more detail elsewhere herein. Thus, the antiviral agent in thecomposition is an antiviral agent which acts in synergy with HCQ (e.g.,as described herein). Optionally, the composition is identified for use(e.g., in or on a packaging material) for use in treating an HCV-relateddisease.

The composition is preferably formulated for administration by a routesuitable for both the HCQ and the antiviral agent.

As discussed herein, HCQ is suitable for oral administration.Furthermore, oral administration is a relatively convenient route ofadministration.

Hence, in some embodiments, the composition is formulated for oraladministration. The antiviral agent is preferably selected so as to besuitable for oral administration.

For a given antiviral agent, suitable routes of administration willtypically be known in the art. For example, ribavirin is known to besuitable for oral administration, whereas interferon is not consideredsuitable for oral administration.

In some embodiments, the antiviral agent is a small molecule (e.g., incontrast to interferon). Optionally, the small molecule is characterizedby a molecular weight of less than 1,500 Da, optionally less than 1,000Da, optionally less than 8,000 Da, optionally less than 600 Da, andoptionally less than 400 Da. In general, small molecules areconsiderably more suitable for oral administration than larger molecules(e.g., polymers).

In some embodiments, the composition is identified for use with anadditional antiviral agent. Optionally, co-administration of thecomposition and an additional antiviral agent is for effecting a methodof treatment comprising co-administration of HCQ and at least twoantiviral agents (e.g., as described herein).

In some embodiments, the additional antiviral agent is unsuitable forinclusion in the composition, and as therefore administered separately.The additional antiviral agent may optionally be unsuitable for theroute of administration of the composition, for example, wherein thecomposition is formulated for oral administration, and the additionalantiviral agent is unsuitable for oral administration (e.g., aninterferon). Alternatively or additionally, additional antiviral agentmay optionally be unsuitable for the frequency of administration of thecomposition, for example, wherein the composition is formulated foradministration once per day, and the additional antiviral agent is moresuitable for administration once per week (e.g., a PEGylatedinterferon-α).

The composition may be, for example, in the form of a liquid, asemi-solid (e.g., gel), or solid.

In some embodiments, the composition is in a solid form. Examples ofsolid forms for a composition include, without limitation, a tablet, acapsule (e.g., comprising an encapsulated solid), a caplet, a powder,microspheroids, and granules.

The composition is preferably formulated in accordance with the intendedfrequency of administration of the composition. This, in turn, willdepend on the properties of the active agents. As discussed herein, HCQmay be administered, for example, once per day, but also at otherfrequencies (e.g., twice or thrice a day). Thus, to the intendedfrequency of administration will depend on the properties of theantiviral agent co-formulated with the HCQ.

Thus, for example, in embodiments wherein the antiviral agent issuitable for administration once per day, the composition is optionallyformulated for administration once per day, in embodiments wherein theantiviral agent is suitable for administration twice per day, thecomposition is optionally formulated for administration twice per day,and so forth.

In some embodiments, the antiviral agent may be administered effectivelyat various frequencies (e.g., once per day, twice per day, and thriceper day). Typically, when other factors are equal, it will be moreconvenient for a subject to be administered a composition once per daythan twice per day, more convenient to be administered a compositiontwice per day than more than twice per day, and more convenient to beadministered a composition a constant number of times per day thandifferent numbers of times on different days.

For any given antiviral agent, optimal (e.g., most therapeuticallyeffective and/or most convenient) frequencies of administration of theagent will typically be known to those skilled in the art.

It is to be appreciated that an active agent can be made more suitablefor less frequent administration (e.g., once per day, as is particularlyconvenient, instead of twice or more per day) by formulating acomposition appropriately, for example, by formulating the compositionfor slow release of the active agents therein.

Slow release preparations typically include slow release biodegradablecarriers. Slow release biodegradable carriers are well known in the art.These are materials that may form particles that may capture therein anactive compound(s) and slowly degrade/dissolve under a suitableenvironment (e.g., aqueous, acidic, basic, etc.) and therebydegrade/dissolve in body fluids and release the active compound(s)therein. The particles are preferably nanoparticles (i.e., in thenanometer range, e.g., in the range of about 1 to about 500 nm indiameter, preferably about 50-200 nm in diameter, most preferably about100 nm in diameter).

The rate at which a drug is released is generally dependent on the rateat which the dosage form disintegrates or dissolves. Disintegrationgreatly increases the drug's surface area in contact with GI fluids,thereby promoting drug dissolution and absorption. Disintegrants andother excipients (e.g., diluents, lubricants, surfactants, binders,dispersants) are often added during manufacture to facilitate theseprocesses. Surfactants increase the dissolution rate by increasing thewettability, solubility, and dispersibility of the drug. Disintegrationof solid forms may be retarded by excessive pressure applied during thetableting procedure or by special coatings applied to protect the tabletfrom the digestive processes of the gut. Hydrophobic lubricants (e.g.,magnesium stearate) may bind to the active drug and reduce itsbioavailability.

Dissolution rate determines the availability of the drug for absorption.When slower than absorption, dissolution becomes the rate-limiting step.Overall absorption can be controlled by manipulating the formulation.For example, reducing the particle size increases the drug's surfacearea, thus increasing the rate and extent of GI absorption of a drugwhose absorption is normally limited by slow dissolution. Dissolutionrate is affected by whether the drug is in salt, crystal, or hydrateform.

Oral slow-release forms are often designed to maintain therapeutic drugconcentrations for greater than 12 hours. The absorption rate can becontrolled by coating drug particles with wax or other water-insolublematerial, by embedding the drug in a matrix from which it is releasedslowly during transit through the GI tract, or by complexing the drugwith ion-exchange resins.

Thus, for example, a slow-release formulation in tablet form, may bebased on the use of a hydrophilic polymer which swells in contact withgastrointestinal fluids, to form a gel, which creates a barrier thatenrobes the tablet. The barrier limits physical exchanges between theinside of the tablet and the surrounding medium. As a consequence,intrusion of water towards the tablet matrix and diffusion of drug areslowed down, allowing a controlled slow release of the drug.

Various types of polymers may be used as a matrix for the slow-releaseof drugs, such as polyvinyl chloride, polyethylene polyamides,ethylcellulose, silicone, poly (hydroxyethyl methacrylate), otheracrylic co-polymers, and polyvinylacetate-polyvinyl chloride copolymers.

In some embodiments, the composition is a unit dosage form (e.g., a unitdosage form formulated for oral administration).

The term “unit dosage form”, as used herein, describes physicallydiscrete units to (e.g., in solid form), each unit containing apredetermined quantity of HCQ and antiviral agent calculated to producethe desired therapeutic effect, in association with at least onepharmaceutically acceptable carrier, diluent, excipient, or combinationthereof (e.g., as described herein).

The amount of HCQ and the antiviral agent in the composition areoptionally adjusted so as to provide an appropriate amount of eachactive agent per day (e.g., as described elsewhere herein). Optionally,the amount of HCQ is sufficient to inhibit HCV-induced autophagy (e.g.,as described elsewhere herein) and/or sufficient to sensitize HCV (e.g.,as described elsewhere herein) to the antiviral agent included in thecomposition.

Optionally, each unit dosage form comprising a therapeutically effectiveamount of HCQ, as described hereinabove, suitable for 1 day (e.g., from400 to 2000 mg, from 500 to 1000 mg, etc.). Optionally, such apharmaceutical composition is further identified for administration onceper day.

Alternatively, each unit dosage form comprises one half of atherapeutically effective amount of HCQ, as described hereinabove,suitable for 1 day (e.g., from 200 to 1000 mg, from 250 to 500 mg,etc.). Optionally, such a pharmaceutical composition is furtheridentified for administration twice per day.

The unit dosage forms described herein may be provided together in a kitwhich comprises discrete unit dosage forms described herein, packagedtogether in a packaging material.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Pharmaceutical compositions of embodiments of the present invention maybe manufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with embodiments ofthe present invention thus may be formulated in conventional mannerusing one or more to pharmaceutically acceptable carriers comprisingexcipients and auxiliaries, which facilitate processing of the activeingredients (HCQ and antiviral agents described herein) intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredient(s) of embodiments of the inventionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological saline buffer with or without organic solvents such aspropylene glycol, polyethylene glycol.

For transmucosal administration, penetrants are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the active ingredients can be formulatedreadily by combining the active ingredients described herein withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the active ingredient(s) to be formulated as tablets,pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions,and the like, for oral ingestion by a patient. Pharmacologicalpreparations for oral use can be made using a solid excipient,optionally grinding the resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries if desired, to obtaintablets or dragee cores. Suitable excipients are, in particular, fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;cellulose preparations such as, for example, maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active ingredients.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules to made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients described herein may be dissolved or suspended insuitable liquids, such as fatty oils, liquid paraffin, or liquidpolyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forthe chosen route of administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the active ingredient(s for useaccording to embodiments of the present invention are convenientlydelivered in the form of an aerosol spray presentation (which typicallyincludes powdered, liquefied and/or gaseous carriers) from a pressurizedpack or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the active ingredient(s and a suitable powderbase such as, but not limited to, lactose or starch.

The active ingredients described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active ingredients. Additionally, suspensionsof the active ingredients may be prepared as appropriate oily injectionsuspensions and emulsions (e.g., water-in-oil, oil-in-water orwater-in-oil in oil emulsions). Suitable lipophilic solvents or vehiclesinclude fatty oils such as sesame oil, or synthetic fatty acids esterssuch as ethyl oleate, triglycerides or liposomes. Aqueous injectionsuspensions may contain substances, which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.Optionally, the suspension may also contain suitable stabilizers oragents, which increase the solubility of the active ingredients to allowfor the preparation of highly concentrated solutions.

Alternatively, the active ingredients may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The active ingredients of embodiments of the present invention may alsobe formulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accompanied by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert.

Compositions comprising HCQ and an antiviral agent, as described herein,formulated in a compatible pharmaceutical carrier may also be prepared,packaged in a packaging material, and identified in or on the packagingmaterial, for treatment of an HCV-related disease, as is detailedherein.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

The word “exemplary” is used herein to mean “serving as an example,instance or illustration”. Any embodiment described as “exemplary” isnot necessarily to be construed as preferred or advantageous over otherembodiments and/or to exclude the incorporation of features from otherembodiments.

The word “optionally” is used herein to mean “is provided in someembodiments and not provided in other embodiments”. Any particularembodiment of the invention may include a plurality of “optional”features unless such features conflict.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Materials:

Chloroquine diphosphate was obtained from Sigma; hydroxychloroquinesulfate was obtained from Sigma;

interferon-α (human, PEGylated) (Peginterferon alfa-2b) was obtainedfrom Schering Plough;

ribavirin was obtained from Sigma-Aldrich.

Boceprevir and NM-107 were synthesized as described in the literature.

In Vitro HCV Replicon Model:

Studies on hepatitis C virus (HCV) replication have been greatlyadvanced by the development of cell culture models known as repliconsystems.

The hepatoma cell line Huh7 is a subclone derived from cell line 9-13. AHuh7 cell line which expresses an HCV genotype 1b replicon 1377/NS3-3′(accession no. AJ242652) was created by Lohmann et al. [Lohmann et al.,Science 1999, 285:110-113]. A replicon-containing cell culture,designated GS4.3, was obtained from Dr. Christoph Seeger (Institute forCancer Research, Fox Chase Cancer Center, Philadelphia, Pa.), and wasprepared as described in Zhu et al. [J Virol 2003, 77:9204-9210].

As shown in FIG. 1, the replicon consists of a subgenomic HCV sequencein which the gene unit encoding the HCV structural proteins is replacedby the gene encoding the neomycin phosphotransferase II (NPTII). NPTIIexpression is under the control of the HCV internal ribosome entry site(IRES), whereas the translation of the region that produces HCV proteinsNS3 to NS5 (up to the authentic 3′-UTR) is controlled by theencephalomyocarditis virus (EMCV) internal ribosome entry site (IRES).The NS3 protein cleaves the HCV polyprotein to release mature NS3, NS4A,NS4B, NS5A and NS5B proteins that are required for HCV replication.

This construct is similar to the replicon present in the cell line 9-13and provides stable NPTII expression for the screening of antiviralagents.

Culture and Treatment of Huh7 Cells:

Huh7 cells were maintained at 37° C., in an atmosphere of 5% CO₂, inDMEM (Dulbecco's modified Eagle medium) supplemented with 2 mML-glutamine, non-essential amino acids (NEAA), 10% fetal bovine serum(FBS) and 500 mg/ml geneticin. Cells were sub-divided at a 1:3 or 1:4ratio every 2-3 days. 24 hours prior to the assay, Huh7 cells werecollected, counted, plated in 96-well plates at 7,500 cells/well in 100ml standard maintenance medium, and incubated in the conditions above.To initiate the assay, culture medium was removed, and cells were washedonce with PBS (phosphate buffer saline). For control compounds only, 90ml assay medium to (DMEM with L-glutamine, NEAA, and 10% FBS) was added.

Test compounds were prepared as a 10× stock in assay medium. Serialdilutions of compounds in assay medium were added in a total volume of10 μl, and the plates were then rocked to mix, and incubated asdescribed above for 72 hours.

Human interferon-α (Peginterferon alfa-2b) and ribavirin are hepatitis Cvirus inhibitors that reduce RNA replication, and were included in eachrun as positive control compounds. Positive control compounds were addedin duplicate at two different concentrations, 1 μM and 1 nM forinterferon, and 100 μM and 200 μM for ribavirin, in order to provide lowand high control values.

Quantification of HCV Levels:

HCV RNA levels were measured using TaqMan® RT-PCR. Total cellular RNAwas isolated and amplified by using a RealTime HCV assay (m1000™Automated Sample Preparation System and m2000rt™ instrument for reversetranscription, PCR amplification, and detection/quantitation, AbbottMolecular Inc.), which detects and quantitates HCV genotypes 1-6. Themolecular genotyping method targets the 5′-untranslated (UTR) region ofthe virus genome (see FIG. 1) and is based on an amplification of theviral genome. An internal control, simultaneously amplified by RT-PCR,served to demonstrate that the process proceeded correctly for eachsample. A negative control, low positive control and high positivecontrol were also introduced. Results are reported in InternationalUnits per ml (IU/ml), and 1 IU/ml=4.3 copies/ml. The lower limit ofdetection was 12 IU/ml with ≧95% probability. The dynamic range of theassay extended from 12 to 100,000,000 IU/ml. The EC₅₀ was defined as theconcentration of compound at which the HCV RNA level in the repliconcells was reduced by 50%.

Quantification of Cytotoxicity:

In order to measure any cytotoxic effect, the viabilities of thereplicon cells following 72 hours of treatment with compound weredetermined using an MTS(3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2Htetrazolium inner salt) assay (CellTiter 96® AQueous One Solution CellProliferation Assay; Promega). The CC₅₀ was defined as the concentrationof the compound at which cell viability was reduced by 50%.

Analysis of Antiviral Drug Combinations:

Antiviral assays were performed by treating Huh7 cells with acombination of two compounds, as described hereinabove for Huh7 cells.Each combination of drugs was assayed in triplicate.

The effects of drug combinations were evaluated according to the methoddescribed by Prichard & Shipman [Antiviral Res 1990, 14:181-205]. Thetheoretical additive effect was calculated from the dose-response curvesof individual compounds by the equation Z=X+Y(1−X) (an equation referredto in the art as a Bliss independence model), where X and Y representthe inhibition produced by the individual compounds and Z represents thetheoretical effect produced by the combination of compounds. Theexperimental results were normalized to the theoretical results expectedfor an additive effect (i.e., “Z”), and the theoretical additive surfacewas then subtracted from the actual experimental surface, to obtain ahorizontal surface which represents synergy between the drugs. Thus,when the surface equals the zero plane, the combination is additiverather than synergistic. A surface that lies above the zero plane (e.g.,at least 20%) indicates a synergistic effect of the combination and asurface lower than the zero plane (e.g., below minus 20%) indicatesantagonism between the drugs.

The effects of drug combinations were also evaluated by calculating aCombination Index (CI) for three different drug ratios in a Loeweadditivity model, using CalcuSyn software based on the method describedby Chou & Talalay [Trends Pharmacol Sci 1983, 4:450-454]. CI values of<1, 1, and >1 indicate synergy, an additive effect, and antagonism,respectively.

The effects of drug combinations were also evaluated by standardisobologram analysis, using CalcuSyn software.

Example 1 Effect of Hydroxychloroquine in Combination with Interferon-αon HCV RNA Replication

Huh7 cells were treated with various concentrations (0, 0.22, 0.66, 2, 8and 18 μM) of hydroxychloroquine sulfate (HCQ) in combination withvarious concentrations (0, 0.41, 1.23, 3.7, 11.1, 33.3, 100 and 300IU/ml) of PEGylated to human interferon-α (Peginterferon alfa-2b), asdescribed hereinabove. The levels of HCV RNA were measured by RT-PCR,and the results were analyzed according to Prichard-Shipman,Chou-Talalay and isobologram models, as described hereinabove. Inaddition, cytotoxicity of the tested combinations of HCQ andinterferon-α (IFNα) was determined as described hereinabove.

As shown in FIG. 2, HCQ and IFNα each inhibited HCV RNA replication in adose dependent manner, both alone and in combination.

As shown in FIGS. 3A and 3B, HCQ and IFNα exhibited a synergistic effectin combination, as determined according to a Prichard-Shipman model. Thesynergistic effect was particularly strong for a combination of 3.7IU/ml IFNα and 6 μM HCQ, for which the inhibition of HCV was 30% morethan expected for an additive effect.

Table 1 presents the Combination Index (CI) values for combinations ofhydroxychloroquine (HCQ) and interferon-α (IFNa) calculated according toa Chou-Talalay model.

As shown in Table 1, HCQ and IFNα exhibited a synergistic effect incombination, with the calculated combination index values all beingconsiderably lower than 1.

TABLE 1 IFNα (IU/ml):HCQ (μM) Combination Index ratio ED₅₀ ED₇₅ ED₉₀ 6:10.69 0.73 0.77 2:1 0.45 0.47 0.49 1:2 0.56 0.56 0.56

ED₅₀, ED₇₅ and ED₉₀ represent amount of drug which result in 50%, 75%and 90% inhibition of viral activity.

As shown in FIG. 4, HCQ and IFNα exhibited a synergistic effect incombination, as determined by an isobologram.

The agreement between the various models indicates thathydroxychloroquine and interferon-α exhibit a considerable synergisticeffect when administered in combination.

As shown in FIG. 5, cell viability was not affected by the tested dosesof to IFNa, and was only slightly reduced by the highest tested dose (18μM) of HCQ.

These results indicate that most of the tested combinations of HCQ andIFNα, including combinations which exhibited a particularly strongsynergy, are substantially non-toxic.

For comparison, chloroquine diphosphate was tested in combination withIFNα, using the same procedures as described for HCQ. Chloroquine andIFNα each inhibited HCV RNA replication (data not shown).

As shown in FIG. 6, chloroquine did not exhibit any appreciablesynergistic effect in combination with IFNa, as determined according toa Prichard-Shipman model.

Example 2 Mechanistic Insights

Effect of Hydroxychloroquine on NF-κB Signaling in HCV Replicon Cells:

The Huh7 harboring replicon model was used to assess the effect ofhydroxychloroquine (HCQ) on HCV infection.

In one set of experiments, HCV replicon-Huh-7/neo cells were seeded in96-well plate for 16 hours. Then cells were incubated with severalconcentrations of HCQ for 72 hours. Replication levels of HCV repliconwere determined by quantification of a replicon-borne neomycin geneproduct (NPT II, % NPTII). Data is presented in FIG. 7. Bars representthe mean of duplicate wells expressed as percentages compared to thecontrol cells, errors indicate standard deviation (SD). Differences werecompared for the HCQ-treated conditions to the corresponding untreatedone using a Student t-test. Basically p-values less than 0.05 wereconsidered as statistically significant and are represented by anasterisk (*).

In another set of experiments, Huh7 cells harboring HCV replicon weretreated during 48 hours with 0.5 μM and 1 μM of HCQ Immunoblot assay,performed to detect NS5A and HCV core proteins, showed that similarly toHCQ is able to decrease both HCV core and NS5a protein level in adose-dependent manner, in comparison to the untreated condition, aspresented in FIG. 8. Western blotting of α-actin showed equal loading ofthe samples.

Since NS5A and HCV core proteins have been shown to regulate theexpression and/or the activation of cellular genes, the effect of HCQ onglobal gene expression was evaluated. Huh7 harboring replicon cells weretreated for 6, 12 and 24 hours with 10 μM of HCQ. Each condition wasperformed in duplicate. Total RNA extracted from cells was hybridized toWhole Human Genome Agilent 4×44K for gene expression analysis.

After normalization, annotated genes in each duplicate whose relativeintensity values were higher than background were selected. The numberof modulated genes was determined using filtering according to a FoldChange (FC) based selection: at least a 2 FC at 6 hours, 12 hours and 24hours.

Fold changes were calculated as follows: gene expression normalizedvalue measured in Huh7 harboring replicon cells treated with 10 μM ofHCQ (performed in duplicate) divided by the corresponding value inuntreated cells, at each time point of kinetic 6 hours, 12 hours and 24hours. Fold Changes (FC) cut-off were fixed at 12 hours and 24 hours asFC≧2 for an up-regulation and FC≦0.5 for down-regulation. Data ispresented in Tables 2 and 3.

As shown in Table 2, most of the modulated genes were down-regulated,and the effect was more pronounced at 24 hours of treatment.

As shown in Table 3, among the 23 modulated genes, only 3 are found tobe up-regulated (FC≧2; in bold) while 20 are significantly repressed(FC≦0.5, in italic).

TABLE 2 Number of genes transcriptionally modulated by HCQ treatmentUp-regulated Down-regulated  6 hours / 5 12 hours  7 30 24 hours 23 63

TABLE 3 Mean value of Fold Change Official (replicate) Agilent ProbeGene RefSeq mRNA HCQ HCQ HCQ Name Symbol ID 6 h 12 h 24 h A_23_P161218ANKRD1 NM_014391 0.8 0.3 0.2 A_23_P259071 AREG NM_001657 1.0 0.5 0.3A_23_P253350 C8orf4 NM_020130 0.7 0.3 0.2 A_24_P135319 CGNL1 NM_0328660.9 0.4 0.4 A_23_P19663 CTGF NM_001901 0.8 0.4 0.4 A_23_P7144 CXCL1NM_001511 0.7 0.2 0.2 A_23_P110204 CXCL5 NM_002994 0.8 0.4 0.3A_23_P163402 CYP1A1 NM_000499 1.1 0.2 0.3 A_23_P46429 CYR61 NM_0015540.7 0.4 0.3 A_23_P108751 FHL2 NM_201555 0.8 0.3 0.2 A_23_P64721 GPR109BNM_006018 0.9 0.4 0.4 A_24_P300394 GSTA2 NM_000846 0.9 0.5 0.4A_23_P153320 ICAM1 NM_000201 0.7 0.4 0.4 A_23_P27584 MYADM NM_0010208181.0 0.4 0.4 A_23_P202156 NFKB2 NM_002502 0.8 0.4 0.4 A_23_P127584 NNMTNM_006169 0.8 0.5 0.1 A_23_P315815 NRG1 NM_013961 0.7 0.4 0.2A_23_P338912 PHLDA1 NM_007350 0.7 0.5 0.4 A_23_P55706 RELB NM_006509 0.90.3 0.3 A_23_P51136 RHOB NM_004040 0.8 0.4 0.3 A_23_P367899 EPORNM_000121 1.2 2.2 2.2 A_24_P20327 KLF15 NM_014079 0.8 2.3 3.0A_24_P406664 RAD23B NM_002874 0.8 2.6 3.3

Interestingly, two down-regulated genes encode for RELB and NFKB2transcription factors. Furthermore, several known target genes of theNF-κB pathway were concomitantly found down-regulated: ICAM1, CXCL1,CXCL5 and CYR61/CCN1. Other down-regulated genes such as C8orf4,GPR109B, NRG1, PHLDA1 and RHOB are functionally linked to the apoptoticprocess GO term.

Since NS5A and HCV core protein are known to activate NF-κB, the downregulation of genes encoding related transcriptional factors by the HCQmight reflect a consequence of an inhibition of the viral proteinexpression.

Effect of HCQ Treatment on Gene Expression Modulations Induced by HCV:

Although the HCV replicon model permits to study the intracellularmodulations occurring during the HCV replication steps, this model islimited by the inability to to support HCV infectious particlesproduction, due to an incomplete viral life cycle, and hence does notpermit to study molecular events occurring during the HCV entry step. Inaddition, this model does not allow studying the intracellularmodifications resulting from acute infection.

Thus, in order to the global impact of HCQ on HCV infection, geneexpression profiling of an infectious HCV cell culture (HCVcc) system,which is closer to the natural infection than the replicon model, wasperformed. Gene expression analysis was performed on HCV infected anduninfected cells to study the global impact of infection; and on HCVinfected cells treated with HCQ, to identify the HCQ effect on host geneexpression previously modulated by HCV.

First, gene expression modulations occurring during infection wereanalyzed by comparing uninfected Huh7 cells and JFH1/CsN6A4 infectedHuh7 cells at 6 hours, 24 hours and 48 hours kinetic time pointspost-infection. It is noted that this model is a model forantiviral-resistant HCV genotype.

Genes whose expression is modulated by infection were selected accordingto data filtering on the basis of at least a twofold change ofexpression compared to the uninfected control condition. Percentages ofmodulated genes were calculated in comparison with 10238 genes, thetotal number of meaningful genes (significantly expressed genes abovethe background and annotated by a RefSeq mRNA ID).

The proportion of genes significantly regulated by infection for eachkinetic time point is illustrated in FIG. 9A. At 6 hours, less than 1%of the total number of genes was found modulated (6 down-regulated and15 up-regulated). At 24 hours, about 13% of genes (1319 genes) areregulated, among which about 8% are upregulated and about 5% aredown-regulated. At 48 hours, about 17% of genes (1736 genes) aremodulated by HCV infection, among which about 9% are up-regulated andabout 8% are down-repressed.

Functional analysis of the transcriptional modulations induced by HCVinfection was conducted in parallel by two complementary approaches: aglobal approach using the Functional Annotation Clustering (FAC) fromDAVID database and a text-mining basis approach using the PredictSearchSoftware™.

Global Analysis Using FAC David Tool:

For the global approach performed using DAVID FAC tool, analysis wasfocused on the 1736 genes regulated after 48 hours of infection, amongwhich 895 are up-regulated and 842 are down-regulated. The FAC showedthat several functional annotations are significantly over-representedin this dataset. The most representative functional clusters arepresented on the heatmap represented in FIG. 9B.

Among these enriched functional annotations, focus was made on thefollowing

Gene Ontology annotations: NF-κB, viral replication, endoplasmicreticulum (ER), cell response to stress/ER stress/UPR, lysosome andmacromolecule catabolic process.

Gene expression analysis was performed on JFH1/CsN6A4 infected Huh7cells treated or untreated, for 12 hours up to 48 hours, with 40 μM ofHCQ. For comparison, and in order to reduce the signal-to-noise ratio,gene expression analysis was also performed on chloroquine (CQ) treatedcells, using the same procedures as for HCQ.

Table 4 presents the total number of genes whose expression is modulatedfrom 12 hours up to 48 hours by treatment of both HCQ and CQ, in JFH1infected Huh7 cells. The number of modulated genes was determinedaccording to a Fold Change (FC) based selection. A 2 FC cut-off wasfixed for HCQ and CQ treated conditions, at 12 hours and 48 hours. FC≧2corresponds to an up-regulated gene, FC≦0.5 for a down-regulated gene.

As shown in Table 4, and while considering the gene expressionmodulations in common with HCQ and CQ, it was observed that less than 1%(62 genes) and about 3% (271 genes) of the total number of genes aresignificantly up-regulated and down-modulated, respectively, aftereither 12 hours or 48 hours of treatment.

TABLE 4 Number of genes modulated in JFH1 infected Huh7 cells treated byboth HCQ and CQ Up-regulated Down-regulated 12 h 38 9 48 h 24 262

Expression data corresponding to the 1736 modulated genes by infectionhave also been included in the FAC and the corresponding expressionprofiles are presented on the heatmaps shown in FIG. 9B.

The expression profiles of host genes differentially expressed (at leasta 2 Fold Change, FC, modulation) following 6 hours, 24 hours and up to48 hours post infection of Huh7 cells with JFH1/CsN6A4 viral particleswere characterized using Agilent 4×44K microarray analysis. Expressionprofiles obtained in other conditions tested to were also included:after a 12 hours up to 48 hours treatment with HCQ and CQ. Hierarchicalclustering (HCL) of each cluster of selected genes was performed usingTMev free software. FC in gene expression were calculated by comparinggene expression in JFH1-infected Huh7 to that in uninfected Huh7 cellsat each time point postinfection. FC of these genes were also calculatedby comparing HCQ and CQ treated infected cells to that in untreatedinfected cells at 12 hours and 48 hours kinetic time pointspost-treatment.

Expression profiles are represented in FIG. 9B on a 2FC scale since FCwere log base 2 transformed. Genes shown in red were upregulated, genesshown in green were down-regulated and genes in black were notmodulated.

Each sample expression profiles is represented in column, and annotatedby a color code horizontal bar figuring on the top of each heatmap.Black bar corresponds to infected conditions, blue bar corresponds to CQtreated condition and yellow bar to HCQ treated cells.

Functional annotations of selected genes regulated by JFH1/CsN6A4infection performed using DAVID FAC tool showed several GO annotationsenriched such as: transcriptional regulation, cell response to stressincluded ER stress, apoptosis, cell cycle. Several other annotationswere found significantly enriched in the set of HCV modulated genes suchas viral replication, NF-κB pathway or lipid biosynthetic process. HCQtreatment was shown to counteract most of the expression modulationsinduced in response to HCV infection.

According to the opposite gene expression profiles observed between HCVinfected conditions and either HCQ or CQ treated conditions, it issuggested that HCQ counteracts gene expression modulations occurringduring infection.

Because of the highest number of modulations of gene expression hasoccurred at the 48 hours kinetic time point post-infection, furtheranalysis was performed at this time point.

Thus, to establish the gene regulatory network illustrating the effectof HCQ on to HCV infected cells, further analysis was focused on the HCVregulated genes whose expression is modulated at least by a twofoldchange by a 48 hours treatment of both HCQ and CQ. 118 HCV modulatedgenes were selected, among which 12 are up-regulated (FC≧2) and 106 aredown-regulated (FC≦0.5) by both compounds, as shown in Table 5 and inFIG. 9C.

TABLE 5 Number of genes modulated by both JFH1 infection and HCQ or CQtreatment at 48 h Up-regulated Down-regulated JFH1 Infection Induced 1104 Repressed 11 2

The obtained data suggest that HCQ mainly acts as a transcriptionalinhibitor (in accordance with the data presented in Table 2). Further,it is shown that most of genes whose expression is down-regulated by theHCQ treatment, are found up-regulated by HCV infection and vice versa,genes up-regulated by the HCQ treatment, are found down-regulated by HCVinfection. This finding is in agreement with previous results obtainedwith the global FAC approach.

PredictSearch:

The list of 118 genes whose expression discriminate both infection andHCQ treatment conditions, was submitted to PredictSearch™ software.PredictSearch™ allows identifying correlations between these genes,functional related genes and biological concepts using a text-miningapproach. The text-mining algorithm extracts pertinent data related tothe selection submitted, among all concepts referenced in thebibliographic data available in the NCBI database. In most cases theseadditional genes are not found significantly transcriptionally modulatedin the microarray data, presumably due to (i) gene may not be modulatedin every biological condition considered (herein, in infection andtreatment); (ii) gene may have a delay response comparing to the kinetictime point studied; and/or (iii) expression modulations occur atpost-transcriptional or post-translational level, which could not beobserved in transcriptomic analysis.

The expression profile of the 57 genes selected on the basis ofPredictSearch to analysis is presented in FIG. 10. Expression profilesof genes obtained in each condition tested were included: at each timepoint post-infection and after a 48 hours treatment of infected cellswith either HCQ or CQ or IFN. These genes were found to be involved inpathways such as ER stress response, autophagy signaling, NF-κB and p53signaling pathways.

Each sample expression profiles is represented in column, and annotatedby a color code horizontal bar figuring on the top of each heatmap.Black bar corresponds to infected conditions, blue bar to CQ treatedcondition, the yellow bar to HCQ treated cells and the orange onecorresponds to IFN treated cells.

These results are in agreement with the global FAC analysis describedhereinabove.

Table 6 summarizes the FC measured for these 57 genes in each conditionof interest. A schematic representation of this functional biologicalnetwork is shown in FIG. 11. This network highlights the pathways whichare over-expressed in HCV infected cells while repressed by the HCQtreatment.

TABLE 6 JFH1 48 h of Official Gene RefSeq mRNA Infection antiviraltreatment Symbol ID 6 h 24 h 48 h CQ HCQ IFN ATF2 NM_001880 1.0 1.6 2.50.8 0.7 0.4 ATF3 NM_001040619 2.0 20.5 65.7 0.4 0.1 0.2 ATF4 NM_0016750.9 2.8 2.1 0.5 0.6 0.5 ATF7IP NM_018179 0.8 2.9 3.4 0.6 0.3 0.4 DDIT3NM_004083 0.9 7.5 13.9 0.4 0.3 0.1 DDIT4 NM_019058 1.8 8.1 5.1 0.5 0.30.3 DUSP1 NM_004417 1.9 6.1 20.6 0.3 0.1 0.1 DUSP4 NM_001394 1.1 2.410.8 0.4 0.2 0.2 DUSP8 NM_004420 1.4 5.8 17.3 0.3 0.1 0.1 TRAF2NM_021138 1.0 1.8 2.4 0.6 0.4 0.7 TRAF4 NM_004295 1.0 1.9 2.8 0.4 0.30.7 UBD NM_006398 1.1 2.5 13.9 0.4 0.3 0.8 AMBRA1 NM_017749 1.0 1.2 2.60.7 0.4 0.4 ATG12 NM_004707 1.0 1.4 1.3 1.2 1.1 0.8 ATG2A NM_015104 0.91.8 4.0 0.5 0.2 0.3 ATG4B NM_178326 1.1 0.4 0.8 1.5 1.6 1.3 ATG7NM_006395 0.8 0.9 1.3 0.7 0.7 0.9 BECN1 NM_003766 1.1 1.6 0.8 0.8 0.91.4 FOXO3 NM_001455 1.2 3.9 2.0 0.4 0.2 0.5 GABARAPL1 NM_031412 1.0 1.52.5 0.7 0.4 0.4 MAP1LC3B NM_022818 1.1 1.4 2.7 0.9 0.8 0.4 PIK3C3NM_002647 1.0 1.8 1.8 1.0 0.7 0.5 SQSTM1 NM_003900 1.0 2.2 4.0 0.5 0.50.5 ULK1 NM_003565 1.1 3.3 3.2 0.5 0.3 0.3 BBC3 NM_014417 1.3 7.4 19.80.5 0.1 0.1 BMF NM_001003940 1.1 3.6 4.4 0.3 0.1 0.2 DAPK3 NM_001348 1.02.0 4.8 0.4 0.2 0.3 GADD45A NM_001924 1.2 4.7 7.5 0.5 0.2 0.2 GADD45BNM_015675 1.8 2.4 11.5 0.6 0.2 0.1 GADD45G NM_006705 0.9 1.7 5.2 1.2 0.70.3 HRK NM_003806 1.1 0.7 1.7 0.4 0.4 0.7 JUN NM_002228 1.4 3.6 7.7 0.40.2 0.2 MYC NM_002467 1.2 2.5 9.7 0.5 0.4 0.3 PDRG1 NM_030815 0.9 1.72.2 0.7 0.7 0.6 PRKAA1 NM_206907 0.9 1.5 3.5 0.7 0.6 0.3 SESN2 NM_0314591.4 9.9 17.3 0.4 0.2 0.2 SIRT1 NM_012238 1.2 4.2 2.9 0.8 0.7 0.5 STK11NM_000455 0.9 1.4 1.3 0.6 0.5 0.9 TNFRSF10B NM_003842 1.2 1.8 2.1 0.80.7 0.6 TSC1 NM_000368 1.0 0.5 1.2 1.2 1.5 0.6 TSC2 NM_000548 1.0 1.21.1 0.6 0.4 0.9 BCL3 NM_005178 1.3 2.3 5.4 0.6 0.5 0.4 CXCL5 NM_0029941.1 1.6 3.7 0.8 0.7 1.3 CYP1A1 NM_000499 1.4 1.8 4.0 0.5 0.1 0.4 CYR61NM_001554 1.8 2.5 6.7 0.3 0.2 0.2 ICAM1 NM_000201 0.9 1.2 2.7 0.7 1.22.0 IKBKAP NM_003640 1.1 1.0 0.3 1.2 2.0 1.3 NFKB1 NM_003998 1.0 1.3 1.70.8 1.0 0.8 NFKBIA NM_020529 1.1 1.6 8.7 0.6 0.5 0.3 NFKBIB NM_0010017160.9 1.9 3.4 0.6 0.6 0.4 NFKBIE NM_004556 1.1 2.1 8.1 0.6 0.4 0.3 NFKBIL2NM_013432 1.0 1.1 0.9 0.7 0.4 1.5 NKIRAS1 NM_020345 1.1 2.2 3.4 0.8 0.40.4 NKIRAS2 NM_001001349 0.9 1.1 1.5 0.8 0.6 1.0 NKRF NM_017544 1.0 2.01.3 1.1 1.3 1.0 RELB NM_006509 1.3 3.1 13.6 0.7 0.3 0.4 TANK NM_1334841.0 0.9 0.8 1.3 1.3 1.2

Effect of Hydroxychloroquine on HCV-Induced NF-κB Signaling:

Among the 57 genes selected, 16 genes that are functionally related tothe NF-κB activation or downstream signaling were found: IKBKAP, NFKB1,NFKBIA, NFKBIB, NFKBIE, NFKBIL2, NKIRAS1, NKIRAS2, NKRF, TANK, RELB,CYP1A1 and CYR61. Most of these genes are found up-regulated by HCVinfection.

Among these 16 genes, expression of several genes was found up-regulatedby HCV while clearly repressed (FC≦0.5) by both HCQ and CQ (CYP1A1 andCYR61). Most of the other genes are differentially expressed byinfection and after 48 hours of treatment by HCQ while slightlymodulated by CQ (0.6<FC≦0.8): NFKBIA, NFKBIB, NFKBIE, NKIRAS1 and RELB.Several other genes that were not found significantly modulatedaccording to their expression ratios were included in the networkbecause they belong to this pathway: NFKB1, NFKBIL2, NKIRAS2, NKRF andTANK. In addition, IKBKAP was included according to its expressionprofile: down-regulated by infection while increased by HCQ treatment.BBC3 and MYC which are related to the p53 and the NF-κB signaling werealso found significantly modulated by both conditions: highly induced byinfection while severely repressed by both compounds. Moreover, BBC3,MYC, CYR61 and other genes such as CXCL5, ICAM1 and BCL3, are directtranscriptional targets of the NF-κB transcription factor genes (see,for example, wwwdotbioinfodotlifldotfr/NF-KB/). CXCL5, ICAM1 and BCL3mRNA expression is increased during infection while slightly repressed(CXCL5 and BCL3) or unchanged (ICAM1) by the HCQ treatment.

Previous studies suggested that HCV infection increases the expressionlevel of several NF-κB related genes. It is demonstrated herein that HCQtreatment counteracts this HCV-induced NF-κB pathway, in accordance withthe results on HCV replicon described hereinabove.

Effect of Hydroxychloroquine on HCV-Induced ER Stress Response and theAutophagic Pathway:

Among the initial set of 57 genes, 12 other genes which were found to bepositively modulated by infection while significantly repressed by theHCQ treatment, are functionally related to the ER stress response andthe subsequent UPR activation: ATF2, ATF3, ATF4, ATF7IP, DDIT3, DDIT4,DUSP1, DUSP4, DUSP8, TRAF2, TRAF4, UBD (see, Table 6 and FIGS. 10 and11).

ATF2, ATF3, ATF4 and ATF7IP are members of mammalian activationtranscription factor/cAMP responsive element-binding (CREB) proteinfamily. Transcription and translation of these ATFs genes is induciblein stress conditions such as ER stress, ATF2 encoded protein, withc-Jun, stimulates the CRE-dependent transcription and directly inducesactivation of DUSP1, 4, and 8 to limit the activities of stress kinasesJNK and p38. In addition, ATF3 is known to be involved in severalpathways such as p53-dependant apoptosis, and T helper cell type (Th)1differentiation activation. ATF4 is the main transcriptional regulatorof the cellular hypoxic response to the Unfolded Protein Response (UPR)and has a key role in the regulation of autophagy in response to ERstress. ATF4 provides a direct mechanistic link between the UPR and theautophagic machinery, probably through DDIT4. Indeed, an increased ATF4expression has been shown to be required and sufficient to upregulateDDIT4.

DDIT4 and DDIT3 are DNA-damage inducible transcripts, activated and alsoup-regulated by ER stress and involved in regulation of autophagy.DDIT3/CHOP, a member of the C/EBP family of transcription factors, isalso involved in apoptosis promotion and in the proinflammatory NF-κB.

DUSP1, 4 and 8 are dual specificity protein phosphatases, which play animportant role in the human cellular response to environmental stress aswell as in the negative regulation of cellular proliferation. Thesegenes negatively regulate members of the mitogen-activated protein (MAP)kinase superfamily (MAPK/ERK, SAPK/JNK, p38), which are associated withcellular proliferation and differentiation. The DUSP4/MPK-2 gene productinactivates ERK1, ERK2 and JNK gene product. The DUSP81MPK-1 geneproduct inactivates SAPK/JNK and p38.

TRAF2 and TRAF4 genes encode proteins members of the TNF receptorassociated factor (TRAF) family. TRAF2 is required forTNF-alpha-mediated activation of MAPKUNK and NF-kappa, functions as amediator of the anti-apoptotic signals from TNF receptors and isinvolved in autophagy. TRAF4 is thought to be involved in the oxidativeactivation of MAPK8/JNK in addition to its role in the regulation ofcell death and NF-kappa B activation. UBD/FAT10 is a TNF-alpha-inducibleubiquitin-like protein with a putative role in immune response. It wasshown to mediate TNF-alpha-induced NF-kappaB activation.

Twelve (12) others genes involved in the autophagic pathway wereincluded in the network (see, Table 6 and FIGS. 10 and 11). Among them,expression of 6 genes is clearly positively regulated by HCV whilerepressed by both HCQ and CQ: ULK1, AMBRA1, ATG2A, GABARAPL1, FOXO3 andSQSTM1. Two other genes among the 18 are less strongly repressed by HCQtreatment: PIK3C3 and MAP1LC3B. The four other genes were also includedin the network according to their known implication in the autophagicpathway, although no significant transcriptional modulation was measuredin both studied conditions: ATG4B, ATG7, ATG12 and BECN1. ATG2A, ATG4B,ATG7 and ATG12 belong to the autophagy related genes (ATGs) family andare involved in the sequential events leading to the activation ofautophagic process. The key genes involved in this process are: ULK1 andKIAA0652/ATG13 for the early phase; UVRAG, MAP1LC3B and SQSTM1/P62 forthe vesicule nucleation step and, ATG12 and GABARAPL1 for the latervesicule expansion and completion step.

MAP1LC3B/LC3B is a marker of the autophagic process activation becauseits expression is induced in several stress conditions such as ER stressand the protein MAP1LC3B is highly expressed on the autophagosomesurface. SQSTM1, a cargo adaptator, interacts with MAP1LC3B to recruitcargo protein which would be digested in the autophagolysosome.

PredictSearch software also permitted to identify FOXO3, a member of theforkhead family of transcription factors, which functions as a triggernot only of apoptosis but also of autophagy. The autophagic role ofFOXO3 in transcriptional induction of genes such as ULK1, ATG13, BECN1,UVRAG/VPS38, PIK3C3, ATG12, GABARAPL1, ATG4B and MAP1LC3B, is known inanother pathological condition. FOXO3 transcription level itself isindirectly regulated by the p53 downstream signaling through the SESN2,STK11, PRKAA1 and SIRT1. According to the level of acetylated SIRT1,FOXO3 expression would be increased.

Previous studies have shown that HCV infection induces an ER stress anda subsequent activation of the UPR, which triggers the activation of theautophagic pathway. Notably, it is demonstrated herein that HCQtreatment counteracts the HCV-induced ER stress response, the UPR andthe autophagic pathway.

Effect of Hydroxychloroquine on HCV-Induced p53 Signaling Pathway:

Among the initial set of 57 genes, 17 p53-target genes were identified:BBC3, TNFRSF10B, GADD45A, GADD45B, GADD45G, DAPK3, HRK, BMF, PDRG1, MYC,SIRT1, SESN2, PRKAA1, STK11, TSC2, TSC1 and JUN (see, Table 6, and FIGS.10 and 11). Expression of all these genes, except for HRK, STK11, TSC2and to TSC1 is up-regulated by HCV infection.

All of these genes, except SIRT1, PRKAA1, TSC1, TNFRSF10B, GADD45G andPDRG1 were also found to be down-regulated by the HCQ treatment. 3 genestranscriptionally regulated by p53 (TSC2, PRKAA1 and SESN2) are closelyrelated to the activation of the NF-κB pathway, to the inhibition of theRheb/mTOR signaling and to the transcriptional activation of FOXO3.STK11, TSC2 and TSC1 were also included in the network according totheir known implication in the p53 pathway, although no significanttranscriptional modulation was measured in both conditions. It isnoteworthy that expression of the p53 transcription factor itself wasnot shown to be modulated by both. However, it is well known that thetranscriptional activity of p53 is mostly regulated by its localizationin nucleus or in cytoplasm.

While previous studies have shown that HCV infection induces p53signaling pathway, it is demonstrated herein that HCQ treatmentcounteracts the HCV-induced p53 pathway.

In summary, the gene regulatory network demonstrated herein illustratesthe molecular events modulated both by HCV infection and by the HCQtreatment. Gene expression analysis of these transcriptional modulationsrevealed that HCQ treatment strongly decreases the HCV-induced NF-κBsignaling, the ER stress, UPR, autophagic signaling and thep53-signaling pathways, suggesting that the HCQ antiviral effect ismediated through the repression of these HCV-induced pathways.

Effect of HCQ on HCV-Induced ER Stress Response, Autophagy, NF-κB andp53 Signaling Pathways:

To determine whether these HCQ inhibitory effects on the HCV-inducedsignaling pathways result from a HCQ direct targeting or are aconsequence of the viral eradication induced by the HCQ effect on thelysosomal pH, further analysis was performed to study the geneexpression modulations induced after a 48 hours treatment withInterferon a (IFN). IFN is an antiviral agent currently used incombination with ribavirin (RBV) as standard therapy against chronic HCVinfection. The molecular mechanisms involved in the antiviral effectinduced by exogenous IFN administration to have been well described.

Gene expression profiling was performed on JFH1/CsN6A4 huh7 cellstreated with 100IU IFN during 48 hours.

As expected, it was shown that exogenous IFN stimulation of HCV infectedcells significantly increased expression of genes involved in the IFNsignaling: CXCL1, CXCL5, EIF2AK2, ICAM1, ICAM2, IF135, IFIT1, IFITM2,IFITM3, ISG20, PML, STAT1, ISG15 and WARS. None of the genes regulatedby IFN were found to be modulated by a 48 hours treatment with HCQ,except IFIT1.

On the basis of the selected 57 genes, the gene expression profilesobtained after IFNα treatment were compared to those previously obtainedafter the HCQ treatment, at the 48 hours kinetic time point. For eachgene of the list, the FC was calculated. The results are presented inTable 6. The expression data corresponding to the IFN treated conditionwere included in the hierarchical clustering presented in FIG. 10.

Similarly to HCQ (and CQ), IFN was shown to repress expression of theHCV-induced genes involved in the following pathways: NF-κB signaling(NFKBIA, NFKBIB, NFKBIE, NKIRAS1, RELB, CY1PA1, CYR61 and BCL3), theER/UPR stress response signaling (ATF2,3,4,7IP, DDIT3,4, DUSP1,4,8), theautophagic pathway (ULK1, AMBRA1, ATG2A, GABARAPL1, FOXO3 and SQSTM1)and the p53 signaling (BBC3, GADD45A, GADD45B, DAPK3, BMF, MYC, JUN, andSESN2).

Several other genes that belong to the set of 57 selected genes werefound differently modulated by IFN and HCQ treatment. These include, forexample, genes involved in the ER/UPR signaling (TRAF2, TRAF4 and UBD),in the autophagy pathway (PIK3C3 and MAP1LC3B), and in the p53 signaling(SIRT1, PRKAA1, STK11, TSC2, GADD45G and HRK). Interestingly, theslightly HCV-induced expression of PIK3C3 (FC:1.8) is repressed by IFN(FC: 0.5) although it is not considerably modulated by the HCQ or CQtreatment (FC-CQ:1, HCQ:0.7). In addition, the HCV-induced expression ofPRKAA1 and MAP1LC3B is clearly reduced by IFN (FC=0.3 and 0.4respectively) while CQ (FC=0.7 and 0.9) and HCQ (FC=0.6 and 0.8) moreslightly reduced them. It can be noted that on the basis of thecalculated expression ratios, HCQ and IFN have a similar greaterrepressive effect than CQ.

In conclusion, it is shown herein that the IFN and the HCQ treatment ofHCV infected cells have a similar repressive effect on the HCV-inducedpathways (ER stress, UPR, NFKB, autophagy, p53 signalings),demonstrating that the HCQ inhibitory effect observed on theseHCV-induced biological pathways is a consequence of the viral reductionoccurring in response to an upstream antiviral direct effect of HCQ (orIFN).

qRT-PCR and Nanostring Validation of Selected Host Gene Expression:

An mRNA quantification using a SYBR-Green qRT-PCR approach and using

NanoString technology was performed in order to validate the dataobtained through microarray analysis. The expression level for severalkey genes of the network (see, FIG. 10) was investigated in the RNAextracted at the 48 hours kinetic point and at each followingconditions: infected cells (Hi/H), cells treated with CQ (HiCQ/Hi) orwith HCQ (HiCQd/Hi) or with IFN(HiIFN/Hi).

SYBRGreen qRT-PCR and Nanostring quantification were performed accordingto common procedures. Ratios were calculated on the basis of relativesignal of a given gene normalized to RPL19 and RPL0 in case of RT-PCR.Nanostring signal normalization was performed using expression measuredfor additional 3 other genes (CLTC, POLR1B and RPL19). Expression ratioswere log base2 transformed therefore a positive value corresponds to aninduction ratio while a negative value to a repression ratio.

The obtained data is presented in FIG. 14, and further supports that themicroarrays, the RT-qPCR and the Nanostring experiments demonstratedreproducible gene expression patterns, although higher FC values wereobtained with Nanostring for most of the genes tested.

The microarray expression of the HCV induced NF-κB related gene CYR61was confirmed by Nanostring. In addition, RT-PCR and NanoString analysesconfirmed microarray relative expression ratios measured for autophagicmarkers such as ULK1, and MAP1LC3B/LC3. Expression of key genes thattrigger autophagy (FOXO3 and SIRT1) was also validated, except forPRKAA1.

While there is a discordance between the microarray result and bothRT-PCR and Nanostring ratios determined for the PRKAA1 mRNA in theinfected condition, IT can be suggested that PRKAA1 expression is ratherrepressed than induced by infection.

The HCQ inhibitory effect on the HCV induced genes involved in the p53signaling (BBC3 and SESN2) was also confirmed. Finally, the microarrayexpression ratios of genes involved in the IFN signaling (ISG15, STAT1,and WARS) were also confirmed by Nanostring technology.

Example 3 Effect of Hydroxychloroquine in Combination with Polymeraseand Protease Inhibitors On HCV RNA Replication

Huh7 cells were treated with various concentrations (0, 0.22, 0.66, 2, 6and 18 μM) of hydroxychloroquine sulfate (HCQ) in combination withvarious concentrations of the antiviral agents NM-107 and boceprevir, inorder to determine whether the combinations exhibit synergy. Theprocedures were similar to those described in Example 1 for HCQ+IFNcombinations.

NM-107 is a viral polymerase inhibitor. It is more commonly utilized ina prodrug form (valopicitabine is a prodrug of NM-107) than as a drug.Boceprevir is a viral protease inhibitor. Thus, these two compoundsrepresent different families of antiviral agents, both in respect to oneanother and in respect to IFN.

0, 0.11, 0.33, 1, 3 and 9 μM NM-107 was combined with HCQ, as was 0,0.041, 0.123, 0.37, 1.1, 3.3 and 10 μM boceprevir. HCV RNA were measuredby RT-PCR, and the results were analyzed according to a Prichard-Shipmanmodel, as described hereinabove.

As shown in FIGS. 13A and 13B, HCQ and NM-107 exhibited a synergisticeffect in combination. The synergistic effect was strongest forcombinations of approximately 0.33 to 1 μM NM-107 and approximately 2 to6 μM HCQ, for which the inhibition of HCV was over 20% more thanexpected for an additive effect.

As shown in FIGS. 14A and 14B, HCQ and boceprevir exhibited a modestsynergistic effect for a portion of the tested concentration ranges, andan additive effect for other concentration ranges. The synergisticeffect was strongest for combinations of approximately 0.37 to 1.1 μMboceprevir and approximately 6 μM HCQ.

Taken in combination with the results presented in Example 1, theseresults indicate that HCQ exhibits a synergistic antiviral effect with abroad variety of antiviral agents. The results further indicate thatsynergistic effects with different antiviral agents are strongest atsimilar HCQ concentrations (e.g., approximately 6 μM HCQ).

Example 4 The Effect of Administering HCQ Combined with PEG-IFN/RBV toHCV Patients Being Non-Responsive to Prior Treatment with PEG-IFN/RBV

The backbone of HCV treatment (Standard of Care; SoC) is PEGylatedinterferon-α-2a (PEG-IFN) and ribavirin (RBV) [M. Stefan et al., 2012.Short Guide to Hepatitis C, 12th Ed., Flying Publisher]. A clinicaltrial was performed in order to assess the add-on effect ofhydroxychloroquine sulfate (HCQ) when combined with SoC on chronic HCVpatients being non-responsive to a previous SoC treatment.

Patient Enrolment Criteria:

Chronic genotype 1b HCV infected patients being non-responsive to SoC(i.e., patients who failed to achieve a sustained virological response(SVR) after treatment with PEG-IFN/RBV) were enrolled.

Responsiveness to prior treatment was determined by documentation ofprior response. Non-responsive patients were categorized into twogroups: (i) patients having less than 2 log₁₀ unit reduction in HCV RNAlevel (IU/mL), compared to baseline at week 12 of the previousPEG-IFN/RBV treatment, were considered as “null responders”; (ii)patients having at least 2 log₁₀ unit reduction in HCV RNA level(IU/mL), compared to baseline at week 12 of the previous PEG-IFN/RBVtreatment, but not achieving an undetectable HCV RNA level (<50 IU/mL)at the end of the previous PEG-IFN/RBV treatment (i.e., not achieving aSustained Virological Response), were considered as “partialresponders”.

Exclusion criteria included hypersensitivity to any one of the threedrugs (HCQ, PEG-IFN, RBV); anaemia, thrombocytopenia, elevated bilirubinlevels (>2.5 mg/dL), elevated ALT and/or AST (>10× upper limit ofnormal), or elevated creatinine (>1.5 mg/dL) and INR greater than 1.5;concomitant liver disease other than hepatitis C; decompensatedcirrhosis; hepatocellular carcinoma (e.g., as determined by suggestiveimaging study or alpha-fetoprotein (AFP) levels of >50 ng/ml); humanimmunodeficiency virus co-infection; major uncontrolled psychiatricillness; active illicit drug or alcohol abuse; serious co-morbidconditions; immunosuppressive treatment including corticosteroids;untreated or uncontrolled thyroid disease; solid to transplant organ(renal, heart, or lung); and pregnancy or unwillingness to practicedouble contraception or abstinence.

Liver Disease Severity Assessment:

Liver disease severity was determined on liver biopsy using FibroTest.Fibrosis was scored on a 5-point scale from 0 to 4, as follows: F0=nofibrosis; F1=portal fibrosis without septa; F2=portal fibrosis with fewsepta; F3=numerous septa without cirrhosis; F4=cirrhosis. Activity ofinflammation was scored on a 4-point scale from A0 to A3, as follows:A0=no activity; A1=mild activity; A2=moderate activity; A3=severeactivity.

HCV RNA Level Measurement:

Plasma HCV RNA level (IU/mL) was measured using standard quantitativeRT-PCR as described hereinabove. HCV RNA measurements were taken on theday of treatment initiation (baseline), four weeks after treatmentinitiation and twelve weeks after treatment initiation.

Treatment Regimen:

HCQ was administered as an oral tablet containing 200 mghydroxychloroquine sulfate (Plaquenil®; Sanofi-Aventis, USA Ltd.) twiceper day (400 mg in total per day).

PEG-IFN (Pegasys®; Hoffmann-La Roche Ltd.) was injected subcutaneouslyonce weekly at a dose of 180 μg (0.5 ml).

RBV (Copegus®; Hoffmann-La Roche Ltd.) was administered as an oraltablet containing 200 mg ribavirin at a daily dose of 1000-1200 mg basedon body weight. If body weight was <75 kg, the total daily dose of RBVwas 1,000 mg, administered as 400 mg (2 tablets of 200 mg, morningintake) and 600 mg (3 tablets of 200 mg, evening intake). If body weightwas >75 kg, the total daily dose was 1,200 mg administered as twice 600mg (3 tablets of 200 mg per intake, morning and evening).

Data Collection and Analysis:

HCV RNA level (IU/ml) was measured at treatment initiation (baseline),at week 4 after treatment initiation and at week 12 after treatmentinitiation.

Efficacy Assessment:

The efficacy of the treatment (HCQ combined with PEG-IFN/RBV on chronicHCV patients who failed to respond to previous PEG-IFN/RBV treatment wasdetermined by the changes (log decline) in HCV RNA levels.

Results:

As shown in Table 7 below, three out of five non-responsive patients(HCV-infected patients who did not achieve a Sustained VirologicalResponse (SVR) from previous standard-of-care treatment) that weretreated with HCQ combined with PEG-IFN/RBV exhibited a greater than 2log₁₀ reduction in HCV RNA level from the baseline after 12 weeks oftreatment (i.e., achieved an Early Virological Response; EVR), which isa positive predictor of a cure.

TABLE 7 Effect of HCQ combined with PEG-IFN and RBV on reducing the HCVlevel in chronic genotype 1 HCV patients being non-responsive to aprevious PEG-IFN/RBV treatment HCV RNA Level Change in HCV RNA Patientlevel Pa- Patient Response to at Pa- tient Liver Previous Week 12 tientGen- Disease SoC¹ Baseline Week 12 (log₁₀ Age der Severity Treatment(IU/ml) (IU/ml) reduction) 29 F F1; A0 Null² 1,281,085 284,329 0.65 46 MF4: A3 Partial³ 5,539,535 4,721 3.07* 60 F F2: A2 Null² 734,270 46 4.20*34 M F4 Null² 654,724 790 2.92* 70 M F4 Null² 1,558,394 26,608 1.77*Log₁₀ reduction of ≧2 compared to baseline. ¹SoC = Standard-of-Care;PEG-IFN/RBV. ²Null response: having less than 2 log₁₀ reduction in HCVRNA level (IU/mL) compared to baseline at week 12 of the previousPEG-IFN/RBV treatment. [Patients who are null responders to SoCPEG-IFN/RBV combination therapy have demonstrated sustained virologicalresponse (SVRs) ranging between 5% and 16% with an optimized PEG-IFN/RBVretreatment (M. Stefan et al., 2012. Short Guide to Hepatitis C, 12^(th)Edition, Flying Publisher)]. ³Partial response: having at least 2 log₁₀reduction in HCV RNA level (IU/mL) compared to baseline at week 12 ofthe previous PEG-IFN/RBV treatment, but not achieving an undetectableHCV RNA level (<50 IU/mL) at the end of treatment. (Patients who arepartial-responders to SoC PEG-IFN/RBV combination therapy havedemonstrated SVRs ranging between 7% and 15% with a standard PEG-IFN/RBVretreatment [M. Stefan et al., 2012. Short Guide to Hepatitis C, 12thEdition, Flying Publisher]).

These results indicate that HCQ is capable of potentiating the activityof anti-HCV agents in the treatment of HCV-infected patients whichfailed to respond to a standard of care treatment.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A method of treating an HCV infection, the methodcomprising: (a) identifying an HCV-infected subject non-responsive to ananti-HCV therapy; and (b) administering to said HCV-infected subject atherapeutically effective amount of hydroxychloroquine or apharmaceutically acceptable salt thereof, thereby treating the HCVinfection.
 2. The method of claim 1, wherein said anti-HCV therapycomprises a treatment with PEGylated interferon α-2a or PEGylatedinterferon α-2b, in combination with ribavirin.
 3. The method of claim1, wherein said HCV-infected subject is lacking a sustained virologicalresponse (SVR).
 4. The method of claim 1, wherein said pharmaceuticallyacceptable salt is hydroxychloroquine sulfate.
 5. The method of claim 1,wherein said therapeutically effective amount of hydroxychloroquine or apharmaceutically acceptable salt thereof is in a range of from about 400to about 2,000 mg per day.
 6. The method of claim 5, wherein saidtherapeutically effective amount of hydroxychloroquine or apharmaceutically acceptable salt thereof is in a range of from about 500to about 1000 mg per day.
 7. The method of claim 1, further comprisingadministering to said HCV-infected subject a therapeutically effectiveamount of at least one antiviral agent.
 8. The method of claim 7,wherein said at least one antiviral agent is selected from the groupconsisting of ribavirin, a viral protease inhibitor, a viral polymeraseinhibitor, an NS4A inhibitor and an NS5A inhibitor.
 9. The method ofclaim 8, further comprising administering to said HCV-infected subject atherapeutically effective amount of an interferon.
 10. The method ofclaim 9, wherein said interferon is PEGylated interferon α-2a.
 11. Themethod of claim 1, wherein said HCV-infected subject is infected bygenotype 1 HCV.
 12. The method of claim 11, wherein said HCV-infectedsubject is infected by genotype 1b HCV.
 13. A pharmaceutical compositioncomprising hydroxychloroquine or a pharmaceutically acceptable saltthereof, an antiviral agent, and a pharmaceutically acceptable carrier,the composition being identified for use in the treatment of a hepatitisC virus (HCV) infection in an HCV-infected subject non-responsive to ananti-HCV therapy, wherein said antiviral agent is ribavirin.
 14. Thecomposition of claim 13, being formulated for oral administration. 15.The composition of claim 14, being in a solid form.
 16. The compositionof claim 13, being a unit dosage form of the composition.
 17. Thecomposition of claim 13, wherein said anti-HCV therapy comprises atreatment with PEGylated interferon α-2a or PEGylated interferon α-2b,in combination with ribavirin.
 18. The composition of claim 13, whereinsaid pharmaceutically acceptable salt is hydroxychloroquine sulfate. 19.The composition of claim 18, wherein said unit dosage form comprises anamount of said hydroxychloroquine sulfate in a range of from about 400to about 600 mg.